| ObjectiveThe purpose of this study was to investigate the temporal and spatial characteristics in the morphological and functional changes of local mast cell(Mast Cell,MC)in the acupoint sensitization at different time nodes with knee osteoarthritis rat models,and preliminary results clarify the mechanism of cellular molecular level of MC in acupoint sensitization and provide objective experimental support for clinical treatment of knee osteoarthritis by acupuncture and moxibustion.Furthermore,the study can enrich the mechanism of acupoint sensitization related to MC and explain the scientific connotation of acupoint sensitization at molecular level.Methods120 SD male rats were randomly divided into normal(N)group,normal saline(NS)group and model(KOA)group,with 40 rats in each group.Then each group was randomly divided into five groups according to the five time nodes on the 0th,7th,14 th,21st and 28 th day,with 8 rats in each group.After adaptive feeding,group KOA used monoiodide(MIA)to induce knee osteoarthritis model.After anesthesia,the hairs of rats near joints were cleaned,and the joints were flexed to the maximum and then the solution that 3mg MIA was dissolved into 50 L 9% normal saline was injected into the bilateral knee joints cavity,group NS was injected with 50 L 9% normal saline into the bilateral knee joints cavity in the same way,and the group N was not treated.The three groups were raised only and no intervention was done.In the group N,group NS and the group KOA,6 animals were randomly sacrificed on the 7th day of the experiment and the left knee joint of the animal was taken,and the articular cartilage was stained with Safranin-O and Fast Green staining to observe the morphology of the articular cartilage,then the OA score of articular cartilage was scored.6 animals were randomly sacrificed on the 0th,7th,14 th,21st and 28 th day after the start of the experiment in the group N,the group NS and the group KOA.Respectively,with the right Heding point(EX-LE2),Yanglingquan point(GB34)and Weizhong point(BL40)as the center,specimens of 1.5 mm × 1.5 mm × 1.5 mm in size(including skin and subcutaneous connective tissue)were taken,using toluidine blue staining technique to observe changes in MC morphology and function.6 non-repetitive fields were randomly selected from each slice,analyzing and recording the number of MC and degranulation changes,calculating the rate of degranulation.In the group N,the group NS and the group KOA,2 rats were randomly selected on the 0th,7th,14 th,21th,and 28 th day of the experiment to be perfused and taken the location of the acupoints.The immunofluorescence(IF)staining technique was used to observe co-expression of mast cell tryptase(MCT)and 5-hydroxytryptamin(5-HT)and histamine(HA)in the tissues of EX-LE2 and GB34 and BL40 at different time nodes.Results1.The results of the Safranine-O and Fast Green staining showed that the articular cartilage surface of the group N and the group NS was smooth and continuous without damage,while the morphology of the articular cartilage in the group KOA was significantly changed.The surface of the articular cartilage was discontinuous,and the matrix was damaged or missing,broken,deprivation,and even cartilage tissue is eroded,the typical pathological changes in the joint surface contour.The OA score of articular cartilage in the group KOA was significantly higher than those in the group N and group NS(P<0.05).2.The results of toluidine blue staining showed that there was no significant difference in the number of MC and degranulation rate of the three points among the group N and group NS(P>0.05).The number of MC and the rate of degranulation of EX-LE2 and GB34 in group KOA were significantly higher than those of the group N and the group NS(P<0.05)at the same time node.The results of the group KOA showed the number of MC and degranulation rate from the 7th to 28 th day at EX-LE2 and GB34 were higher than 0 days.There were significant differences in the number of MC and degranulation rate at different time nodes(P<0.05).3.The results of IF staining showed that co-expression of MCT and 5-HT and HA in the group KOA of EX-LE2 and GB34 was co-expressed,but there was no or less co-expression at BL40;the co-expression phenomenon did not occur in the group N and group NS.Conclusions1.Injection of MIA into the articular cavity can cause obvious pathological changes in articular cartilage,and this method can successfully induce a knee osteoarthritis model.2.The MC in the acupoint sensitization presented typical recruitment and degranulation changes and the characteristics of the temporal and spatial changes of MC occured in EX-LE2 and GB34 at different time nodes;acupoint sensitization is a dynamic process.Sensitization takes place from a certain time node and continues for a period of time.Acupoint sensitization has the characteristics of temporal and spatial changes.3.The substances released from the degranulation of MC in acupoint sensitization are 5-HT and HA.These two substances have different expression at different time nodes of acupoint sensitization.The mechanism of MC and 5-HT,HA change in acupoint sensitization may be that the release of 5-HT,HA and MCT may directly affect the circulation of affected acupuncture points,nerves and immune networks,which play a triggering role in acupoint sensitization. |
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