| BackgroundThe function of acupoints are the core problem of acupuncture and moxibustion field.Clinical and experimental studies have observed that when visceral lesions occur,the relevant acupoints on the surface of human body or experimental animal are sensitized,and the local physical and chemical properties will change accordingly,resulting in neurogenic inflammatory reaction and the formation of "sensitization pool".During sensitization,acupoints are from "silent"(physiological status)to "active"(pathological status),the size of the acupoints increases,and their functions of reflection and treatment of the disease are enhanced,and the phenomenon of "acupoint sensitization" appears.The sensitized acupoints on the body surface caused by viscera lesions mostly appear in the same or adjacent nerve segments,and sensitization sites are often accompanied with paresthesia or histopathological changes and other sensitization phenomena.Besides the in-depth studies on the sensitized acupoints,the enhanced function of sensitized acupoints become a key issue to be clarified.Here in,when acupoints are in"active state",the enhanced effect of acupuncture inputs on multi-level of the central nervous system(CNS)deserves further exploration.Several studies have shown that vagus nerves play a key role in alleviating inflammatory injury,while electroacupuncture(EA)at Zusanli(ST-36)can alleviate systematic inflammation in lipopolysaccharide(LPS)-peritonitis and regulate inflammatory factors in breast tumor mice,via activating vagus nerve outputs.These results indicate that EA may exert systemic anti-inflammatory effect by activating the vagus nerve.Therefore,when visceral inflammatory lesions occur,does EA at sensitized acupoints mediate a more effective anti-inflammatory effect by enhancing its influence on related neurons in CNS?In the present study,we sought to firstly identify the distribution pattern of sensitized acupoints in colitis rats induced by dextran sulfate sodium salt(DSS).Secondly,we observed the ameliorating effect of EA at sensitized acupoints on inflammatory injury in colitis rats.The effects of EA on neurons in superficial(Ⅰ-Ⅱ)and deep(Ⅲ-Ⅴ)laminae of spinal dorsal horn and choline acetyltransferase positive(ChAT+)neurons of brainstem dorsal motor nucleus of vagus(DMV)between the sensitized acupoints and the same acupoints in normal condition were compared.The purpose of this study is to provide an experimental basis for clarifying the changes of function following acupoint sensitization.Objective(1)To clarify the improvement effect of EA at sensitized acupoints on inflammatory injury in colitis rats(2)To observe whether the functional activities of neurons in spinal dorsal horn and ChAT+ neurons in DMV were involved in the process of EA at sensitized acupoints alleviating colon inflammationMethodsThis experimental study is mainly divided into three parts:(1)To determine the sensitized acupoints of colitis rats:male SD rats were randomly divided into normal group and model group.During the experiment,rats in model group were administered with 5%dextran sulfate sodium(DSS)solution for 6-day-free drinking to induce the colitis model,and rats in the normal group were given water for drinking.At day 7(the day after successful modeling),Evans blue(EB)solution was injected into the rats in both groups via caudal vein.Two hours later,according to the locations of EB exudation areas on the body surface of colitis rats,the sensitized acupoints were determined for further study.(2)To observe the effect of EA at sensitized acupoints on colonic inflammatory injury:based on the results in Part(1),SD rats were randomly divided into normal group,model group and EA group.The modeling method was the same as above.At day 7,under isoflurane anesthesia,EA stimulation(2 mA,2 Hz/15 Hz,30 min/d,continuous 6 d)was applied at sensitized acupoints in EA group.Normal group and model group were anesthetized in the same way,but no other intervention was applied.At day 0,7 and 13(the day after the end of EA),the effects of EA at sensitized acupoints on disease activity index(DAI)scores,the mechanical paw withdrawal threshold and thermal paw withdrawal latency were determined in the normal group,model group and EA group rats.DAI scores are the mean of stool consistency scores and bleeding scores.The colon of rats was stained with HE to observe the histological injury of the colon,and the histological tissue damage of the colon was scored.(3)To clarify the effects of EA at sensitized acupoints on the activity of neurons in spinal dorsal horn,as well as ChAT+neurons in DMV of colitis rats:SD rats were randomly divided into four groups:normal group,normal+EA group,model group and EA group.The modeling method was the same as above,and rats in the normal+EA group were given water for drinking.At day 7,under isoflurane anesthesia,EA stimulation(2 mA,2 Hz/15 Hz,30 min,once)was applied at sensitized acupoints in normal+EA group and EA group.The spinal cord segments corresponding to the sensitized acupoints and the brain stem of rats was collected for immunofluorescence staining of c-Fos protein(spinal cord and DMV)or ChAT protein(DMV).Normal group and model group were anesthetized in the same way,but no other intervention was applied.The number of rats in each group above was not less than 5.Results1 Determination of sensitization acupoints in colitis ratsAt day 7(the day after successful modeling),after EB was injected through caudal vein,there were more EB exudation points on the body surface of rats in the model group,which were mainly distributed in T12～S1 segments,including the surface areas of lower extremities,lower back and lower abdomen,while mostly concentrated in L2(24.4%11/45)and L5(20%9/45)segments.Clinical research reported that the most commonly used acupoints for colitis are:Zusanli(L4),Tianshu(T11),Zhongwan(T10)and Shangjuxu(L5).According to the distribution pattern observed in our study,bilateral Shangjuxu(ST37,L5)were chosen as the sensitized acupoints for further investigation.There were no or only a small amount of EB extravasation points on the body surface of normal rats,suggesting sensitization occurred in the Shangjuxu acupoints area in colitis rats.2 The ameliorating effect of EA at sensitized acupoints on inflammatory injury in colitis ratsAt day 7,the mechanical paw withdrawal threshold and thermal paw withdrawal latency in the model group and the EA group were significantly lower than those in the normal group(P<0.001).The stool was loosing,and there was obvious blood in the stool and perianal area.The DAI score and histological tissue damage scores of the colon in the model group and the EA group were significantly higher.At day 13(the day after the end of EA),the mechanical paw withdrawal threshold in the model group was still significantly lower than that in the normal group(P<0.001);However,the mechanical paw withdrawal threshold in EA group was higher than that in model group(P<0.001);DAI score and histological scores of colon were lower than those of model group(P<0.01;P<0.05).These results suggested that EA at sensitized acupoint(ST37)could significantly improve the inflammatory injury of colitis rats.3 Effects of EA at sensitized acupoints on the activity of neurons in spinal dorsal horn and ChAT+neurons in DMV of colitis ratsAt day 7,after 30 min-EA stimulation,neurons in superficial laminae(Ⅰ～Ⅱ)of L4-L6 spinal dorsal horn of normal+EA group were significantly increased compared with that of normal group(P<0.05);neurons in superficial laminae(Ⅰ～Ⅱ)of L4-L6 spinal dorsal horn of the model group were significantly increased compared with that of the normal group(P<0.01);Compared with the normal+EA group and the model group,neurons in superficial laminae(Ⅰ～Ⅱ)of L4～L6 spinal dorsal horn of EA group were significantly increased(P<0.001).At day 7,after 30 min-EA stimulation,ChAT+ neurons in DMV of normal+EA group were significantly increased compared with that of normal group(P<0.05);ChAT+neurons in DMV of the model group were significantly increased compared with that of the normal group(P<0.01);Compared with the normal+EA group and the model group,ChAT+neurons in DMV of EA group were significantly increased(P<0.001).These results suggested that both inflammatory colonic injury and EA stimulation can increase the activity of neurons in the superficial laminar of spinal dorsal horn in normal rats,as well as ChAT+ neurons in DMV.In colitis rats,however,EA at sensitized acupoints can produce more enhanced activity of neurons in the superficial laminae of spinal dorsal horn and ChAT+neurons of DMV in rats.Conclusions(1)The sensitization points of the colitis rats were concentrated in the L2 and L5 nerve segments,and sensitization occurred in the Shangjuxu(ST37,L5)acupoints area.At the same time,the colitis rats presented plantar pain sensitivity.(2)EA at sensitized acupoints can improve inflammatory injury and plantar mechanical hyperalgesia in colitis rats.(3)EA at sensitized acupoints can cause the enhancement of sensory afferent and vagal efferent activities in rats,which might mediate their alleviating effect on colitis. |
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