| According to the meridian doctrine of traditional Chinese medicine,assembled meridians are gathered in ear,all the six kinds of yang meridians can directly or indirectly go upward preauricula or aurimidst,six kinds of YIN meridians can indirectly correlate with ear by the collaterals of yang meridians.All the physiological or pathological action of entrails can reflect in ear by meridian.Zusanli is the He point, and also is lower He point,"concentrating the disease in internal organs" is the first acupoint selection method,and possesses of regulating the stomach and invigorating the spleen and calming the adverse-rising energy.Considerable clinic and animal experiment all confirm that there is manifest therapeutic effect of acupuncture "Erwei" acupoint and "Zusanli" acupoint in gastric ulcer.But the effective mechanism hasn't made clear up to day,to result in affecting the effectiveness of prevention and cure.Objective:To study the dependability between the therapeutic efficacy of electroacupunce(EA) of "Erwei" acupoint and "Zusanli" acupoint in experimental gastric ulcer rats and mast cells(MCs) and peritoneal MCs(PMCs) from their metergasis.Methods:The Wistar rats(female and male rats each amounted a half) were used in all the experiments,and they are provided by experimental animal center of Chinese Academy of Medical Sciences.①Morphology and electrophysiology experiment:The 40 Wistar rats were randomly divided into normal control group,model group,electroacupuncture(EA) group and model-electric conduction group(this group was abbreviated to electric conduction group).Experimental gastric ulcer model was established by injecting 0.05ml 20%glacial acetic acid into anterior wall between muscular layer and Serous layer in the gastric antrum under anesthesia for the rats of the last 3 groups. Then,both sides "Erwei" acupoint(CO4) and "Zusanli" acupoint(ST36)were selected and stimulated by electroacupuncture with 2HZ only in the EA group,we firstly determined once Ampere value at "Erwei" acupoint(CO4) before electroacupuncture, 2 hours later,we determined another Ampere value at "Erwei" acupoint(CO4), afterwards,we determined once Ampere value per day,until the 7th day after gastric ulcer operation.The rats of electric conduction group only determined Ampere value at "Erwei" acupoint(CO4) simultaneously with the EA group every day.7 days later, the tissues of the points of the 4 group rats were stained with toluidine bule(TB) after stained with acetylcholine esterase(AchE);while the gastric ulcer tissues were stained with hemotoxylin-eosin(HE).②PMCs collection and culture and flow cytometry detection:a PMCs collection and centrifugalization:we collected rinse solution of peritoneal cavity of 40 Wistar rats in the sterility condition,then,4℃,1000r/min centrifuged 10min,and got the cell suspension and added it into PMCs isolation solution,4℃,2500r/min centrifuged 15min,and got the PMCs at the bottom,which were washed twice with cold PBS without Ca2+,Mg2+(4℃,1000r/min centrifuged 10min),and quit the clear supematant liquid.b PMCs culture and serum preparation:we got the PMCs at the bottom,and put the PMCs into the RPMI-1640 incubation fluid with 10%calf serum(stained with TB, and counted the cell number by light microscope,and regulated the PMCs density at 106piece/ml,and the PMCs purity over 95%;stained with trypan bule,and kept the PMCs vital force over 95%).The 30 Wistar rats were randomly divided into normal control group,model group and EA group(we handled the homologous model according to the method in the morphology experiment for the rats of the 3 groups,but the rats of the EA group didn't determine Ampere value at "Erwei" acupoint(CO4) before electroacupuncture), after 7 days we respectively got the blood from carotid artery of the 3 group(normal control group,model group and EA group) rats,and shifted it in centrifuge tube,37℃stood 2 hours,2500r/min centrifuged 10min,took serums with measuring pipette,and mixed them respectively with the same group,and filtrated sterilization.c PMCs vitro degranulation experiment and flow cytomtry analysis:We respectively added the 3 group serums into the above-mentioned stood-by PMCs suspension(the ratio between PMCs suspension and the serums was 1:1),and incubated 2 hours in 37℃5%CO2 incubator and terminated reaction at 4℃.We washed 106 PMCs with PBS twice(4℃,1000r/min centrifuged 5min),and suspended the cell pellet in 100μl of Annexin-V-Fluos labeling solution,and added 100μl Annexin-V-Fluos labelling solution(prediluted 20μl Annexin-V-Fluos labeling reagent in 1ml incubation buffer and added 20μl Propidium iodide solution),and incubated 10-15min at 15-25℃,and analyzed on a flow cytometer.Results:①Morphology experiment:The MCs in the two acupoints from the dermis to subcutaneous tissues were compared among the 4 group rats,we found that the number of MCs in the model group was the most among the 4 groups,there were remarkable difference(P＜0.01,P＜0.01,P＜0.01),and the number of MCs in the electric conduction gr(?)up was more than the EA group and the normal control group (P＜0.01,P＜0.01 ),and the number of MCs in the EA group was more than the normal control group(P＜0.01).While in the model group,the degranulation ratios of MCs in the two acupoints obviously exceeded than the normal control group and the EA group and electric conduction group,there were remarkable difference(P＜0.01,P＜0.01,P＜0.01),and the degranulation ratios of MCs in the electric conduction group was more than the EA group and the normal control group(P＜0.01,P＜0.01), and the degranulation ratios of MCs in the EA group was more than the normal control group(P＜0.01).Mucous layer in the control group was integrity and without defec;while in the model group,gastric ulcer overtopped lamina muscularis mucosae gastris;space-occupying lesion in the EA group didn't overtop lamina muscularis mucosae gastris,space-occupying lesion in the electric conduction group was between the model group and the EA group.②Electrophysiology experiment:After operation,the electric conduction in the electric conduction group and the EA group was significantly higher than before operation.But the electric conduction in the EA group didn't step up after the first electroacupuncture,and kept up this level after that;while the electric conduction in the electric conduction group continued to step up,and keep this state for 3 or 5 days until the 6th day,then began to descend.The electric conduction of ear acupoint in the two groups reflected the interclass significant difference after the first electroacupuncture,which gradually got close to each other after the 7th day.③MCs collection and culture and flow cytometry detection:In the model serum group,the degranulation ratios of PMCs obviously exceeded than the normal control serum group and the EA serum group,there were remarkable difference(P＜0.01), and the degranulation ratios of PMCs in the EA serum group were more than the normal control serum group(P＜0.01).Conclusions:①MCs in acupoints distribute along nerves and blood vessels,particularly intensive surrounding small vessels,which demonstrates that MCs are really correlated with acupuncture point reaction and acupuncture effect.②The changes of the number and degranulation ratios of MCs offer positive correlation with the occurrence of experimental gastric ulcer.This provides experimental evidence for the theory on body surface related with entrails.③The effect of electroacupuncturing experimental gastric ulcer rats are tightly close to the decrease of the number and degranulation ratios of MCs,and acupuncture serum has the direct repression on degranulated PMCs,which indicates that the mechanism of acupuncture response is relative to some factors,such as nerve, immunity,body fluid and so on.④MCs have some dependability on low resistance in acupoints,which provides the referred scientific witness for clinical application of ear acupoint diagnosing and curing correlated diseases. |
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