| Veterinary acupuncture has a history of more than two thousand years.The Traditional needle techniques such as dry needle,moxibustion and the modern developed techniques such as electroacupuncture,aquacupuncture,laser,acupoint implantation and other therapies have been widely used in animal clinical.Diarrhea is a common and frequent disease in human and animal clinic,while acupuncture is one of the effective therapies for relieving diarrhea.At present,the curative effect of acupuncture on diarrhea has been confirmed and a large number of acupuncture points have been used in clinical practice,however,it is not clear as to which acupoint(s)and which needle technique(s)exerted the best results on the diarrhea.On the other hand,there is a short of the scientific research on the pathway and mechanism of acupuncture actions,and all the available research are in the early stages.The present study was aimed to explore and compare the pathway and mechanism of three acupoints and three needle techniques for treating diarrhea.The three most commonly used acupoints for diarrhea were Hou-san-li(ST-36),Hou-hai(GV-1),Da-chang-shu(BL-25).The three techniques were dry needle(DN),electroacupuncture(EA),and aquacupuncture(AA).Rabbit diarrhea was induced by Fan Xie Ye(Senna)water extracts and the rabbit gastrointestinal(GI)motility and GI ion channels were explored on the rabbit.The goal of the present study is to provide the scientific evidence on the effect and mechanism of using acupuncture on diarrhea,thus has high academic significance and clinical application value.For the rabbit model study,twenty female New Zealand white-eared white rabbits were randomly divided into 4 groups:control group,dosage Ⅰ group,dosage Ⅱ group and dosage Ⅲ group.Except the control group,the rabbits in the other three groups were fed with different concentration of senna leaf water extract(1 g/kg·d,2 g/kg·d,3 g/kg·d)daily for consecutive 7 days,while the rabbits in the control group were fed with the same amount of normal saline under the same conditions.During the experiment,the rabbit mental state was observed,the body weight and feces status were counted,the diarrhea rate,fecal dilution rate,fecal grade and diarrhea index were calculated.After the study,blood was collected from the ear margin vein to collect the serum and plasma,and the levels of gastrin(GAS)and motilin(MTL)were measured by radioimmunoassay.The contents of vasoactive intestinal peptide(VIP)and 5-hydroxy tryptamine(5-HT)in the serum were measured by ELISA.Results showed that compared with the control group,the weight gain of the rabbits in three senna groups was decreased.On the 1st day of the experiment,the diarrhea rate in all three senna groups reached 100%,and the loose stool rate,fecal grade and diarrhea index were significantly higher than those of the control group,which showing a dose dependence manner.On the 7th day of the experiment,the rabbits in the group with senna dosage 1 g/kg·d had no signs of diarrhea,while the rabbits with 2 g and 3 g/kg·d senna still had significant higher diarrhea indexes than the rabbits in control group.The contents of serum GAS and plasma MTL in the three senna dosage groups increased in a dose-dependent manner compared with the control group,and the 2 g and 3 g/kg·d senna groups were significantly different(P<0.01).The VIP content of each senna group was significantly higher than that of the control group(P<0.05 or P<0.01).The 5-HT content in each group was significantly increased as well compared with the control group(P<0.01).It was noted that all three senna dosage(1 g,2g,3g/kg·d)would affect rabbit body weight,mental state and gastrointestinal hormone levels in some degree.Considering the diarrhea stability,the clinical indicators and gastrointestinal hormones level,2 g/kg·d senna extract was chosen as the ideal dosage to induce the rabbit diarrhea for the following studies.In order to explore the mechanism of DN on different points for treating diarrhea,thirty rabbits were randomly divided into 5 groups:control group,diarrhea model group,ST-36 group,GV-1 group,BL-25 group.All the rabbits in diarrhea model group and the three acupoint groups were administered with 2 g/kg·d water extract of senna for consecutive 7 days to induce rabbit diarrhea,while the rabbits in the control group were gavaged with the same amount of physiological saline instead of senna water extract under the same conditions.After administrating the senna,the acupuncture group was treated with DN acupuncture at the acupoint of ST-36,GV-1,BL-25 respectively,and the model group was stimulated in the non-acupoint area on the inner thigh under the same conditions for a period of 7 days.HE staining was used to observe the changes of rabbit intestinal histomorphology in each group;radioimmunoassay was used to detect the levels of GAS and MTL;ELISA was used to detect the contents of VIP and 5-HT;Immunohistochemical test was used to detect the expression of substance P(SP),enterochromaffin cell(EC)and aquaporin4(AQP4)in intestinal histomorphology.Western blot method was used to detect the expression of tryptophan hydroxylase(TPH)and Na+/H+ exchanger 3(NHE3)proteins.Results showed that compared with the model group,the rabbit body weight in the ST-36,GV-1 and BL-25 three groups increased significantly on the 5th and 7th day of acupuncture(P<0.01).DN acupuncture at ST-36 bilaterally could significantly reduced the loose stool rate,fecal grade and diarrhea index on the 1st day(P<0.01),and reached the same level as the blank control group on the 3rd to 7th days.DN at GV-1 also reduced the loose stool rate on the 1st day(P<0.01),and decreased the diarrhea index on the 1st and 7th days(P<0.01).DN at bilateral BL-25 could significantly decrease the loose stool rate,fecal grade and diarrhea index only on the 1st day(P<0.01).In terms of clinical symptoms,ST-36 is the best one to eliminate the diarrhea,followed by GV-1 and BL-25.Intestinal histomorphology study showed that the height of villi in each group increased significantly(P<0.01),and the ratio of villi height to crypt depth increased as well(P<0.01).But there was still slight swelling of intestinal villi in ST-36 and GV-1 groups,while BL-25 villous structure improved a lot.For the GI motility,study showed that ST-36 could significantly reduce the content of GAS,MTL,5-HT,VIP(P<0.05 or P<0.01),GV-1 could significantly reduce the content of MTL and VIP(P<0.05),BL-25 could significantly reduce the content of GAS,MTL and VIP(P<0.05 or P<0.01).For the expression of SP in intestinal,immunohistochemical results showed that GV-1 could be the best point to exert inhibiting effect(P<0.01),followed by ST-36 and BL-25.For the DN effect on the GI ion channels,results showed that all the three points,ST-36,GV-1 and BL-25,could significantly increase the content of intestinal motility factor EC and ion channel AQP4 in tissues(P<0.01),but there was still a significant difference compared with the blank control group(P<0.05 or P<0.01).The expression of TPH in intestinal of the three groups was significantly decreased(P<0.01),while the expression of NHE3 was increased(P<0.01)compared with the diarrhea model group,but the expression of both TPH and NHE3 in the 3 DN groups were not reach the level in that of the blank control group(P<0.01).Based on the above results,DN at ST-36 exerted the best results on relieving the diarrhea among the three acupuncture points.In order to compare and clarify the therapeutic effect of DN,EA and AA on diarrhea,the ST-36 point was selected.Twenty-five rabbits were randomly divided into 5 groups:blank control group,diarrhea model group,DN group,EA group and AA group.Firstly the model group and the three acupuncture groups were administered with the water extract of senna(2 g/kg·d)for 7 days,and then bilateral ST-36 were stimulated with DN,EA and AA respectively for the three acupuncture groups,while the model group was stimulated with non-acupoints on the inner thigh under the same conditions for 7 days.The blank control group was treated the same way as the above experiment.Results showed that compared with the model group,EA at bilateral ST-36 can significantly reduce the loose stool rate and diarrhea index on the 1st day(P<0.01),and reached the same level as the blank control group from the 3rd to 7th days.DN at ST-36 can reduce the loose stool rate and diarrhea index on the 1st and 3rd days(P<0.01),and no loose stools in the following days.AA had a significant effect on the 1st and 3rd days compared with the model group(P<0.05 or P<0.01).The height of the villi in the AA group was significantly improved(P<0.01),and the depth of the crypt was significantly increased(P<0.01),but the height of the villi and the ratio of the height of the villi to the depth of the crypt were not be able to reach the level in the blank control group(P<0.01).In the DN and EA groups,the villi increased significantly(P<0.01),the crypt structure was clear and complete,and the ratio of the height of the villi to the depth of the crypt increased(P<0.01)as well.For the GI motility,DN,EA and AA could significantly reduce the content of GAS(P<0.01),while only EA exerted prominent effect on the content of MTL(P<0.01),and the difference between aquacupuncture and VIP in the control group is very significant(P<0.01).In addition,DN,EA and AA at ST-36 could significantly inhibit the expression of SP in intestinal(P<0.01),and inhibited the expression of TPH(P<0.01)),but significantly increased the content of NHE3(P<0.01).Compared with the blank control group,the difference was very significant(P<0.01).EA could obviously promote the expression of EC and AQP4 in tissues(P<0.01),and the effect reached the level in the blank control group.Conclusion:2 g/kg·d senna water extract for 7 days could be ideal to induce a stable rabbit model of diarrhea.Dry needle stimulation of ST-36,GV-1,BL-25 could alleviate senna-induced diarrhea in rabbits.Among them,ST-36 could reduce the content of gastrointestinal hormones,causing changes in the protein content of intestinal histomorphology,up-regulated the intestinal ion channels,and slowed down GI motility.For BL-25,in addition to down-regulating the level of gastrointestinal hormones and up-regulating the intestinal ionophore,BL-25 could also act on intestinal histomorphology and reduce the swelling and shedding of intestinal villi.The effect of BL-25 is slightly weaker than ST-36 for the overall effect.GV-1 mainly acted on local gastrointestinal nerves,regulated the abnormal gastrointestinal hormone content,increased intestinal ion channels,down-regulated blood VIP content and SP immune cell expression in intestinal histomorphology,and thereby regulated gastrointestinal peristalsis.And according to clinical diarrhea indicators,the post-acupuncture effect of stimulating GV-1 and BL-25 is better than the immediate effect.Among the three acupoint groups,ST-36 had the best effect with DN technique for reliving diarrhea.For the three needle techniques,dry needle,electroacupuncture and aquacupuncture with bilateral ST-36 could relieve the diarrhea,with the best results in electroacupuncture group,which could reach to the level in the blank control group by reducing 5-HT,increasing the expression of EC and AQP4,indicating that electroacupuncture has the strongest and the best effect. |
PDF Full Text Request