Regulating Function Of Electroacupuncture At Zusanli Point In Rats With Gastrointestinal Motility Disorder After Spinal Cord Injury

Aim:1 To investigate regulating effect on electroacupuncture at zusanli point in rats with gastrointestinal motility disorder after spinal cord injury,and to observe possible reasons which bring on gastrointestinal motility disorder primarily.2 By means of examining the change of stomach emptying,intestine propulsion rate, NOS,receptor before and after treatment,to draw a conclusion of possible approach and mechanism of its function.Method:1 The establishment of spinal cord injury(SCI)model:The model of spinal cord injury was established by WD method,adopted a BBB grade method to estimate and regarded zero score in BBB grade as successful model.2 The sport ability of the rats assess:Improved CBS method was adopted to estimate the behavioral grade of animal sport,which included such indexes as the sport ability in open space,stretch of toe,the reflection of touching the gound,the reflection of return,the reflection of turning over and the experiment of inclined slope.The sport function of the hind legs in every group was observed by BBB grade method.3 Grouping:Rats were randomly divided into five groups:normal control group, model group,side acupoint group,electroacupuncture treatment group and Mosapride treatment group.(1)Normal control group:The normal rats were fixed for 30 minutes every day without electroacupuncture for 14 days.(2)Model control group:The rats with spinal cord injury were fixed for30 minutes every day without electroacupuncture for 14 days.(3)Side acupoint group:The rats with spinal cord injury were treated with electroacupuncture at side acupoint near zusanli acupoint for 30 minutes every day for 14 days.(4)Electroacupuncture treatment group:The rats of spinal cord injury were treated with electroacupuncture at zusanli for 30 minutes every day for 14 days.(5)Mosapride treatment group:The rats with spinal cord injury were treated with Mosapride for 30 minutes every day for 14 days.4 Choosing acupuncture point and acupuncture method:According to "animal acupuncture points diagram" specified by experimental acupuncture graduate school of Chinese acupuncture academic association,zusanlli points on both sides of feet were selected(zusanlli point was about 5mm under capitulum fibulae at outer hind knee joint).The sober rats in each electroacupuncture treatment group were fixed, which started to be treated with electroacupuncture after one week's model establishment.It kept stabbing in 7mm,linking electroacupuncture treatment instrument of type of stomach and intestines.The stimulus parameters were continuous wave,3Hz(frequency)and 2-3mA(strength),which led to the rats' leg to tremble slightly for degree.It kept 30 minutes once every day.5 Stomach emptying and intestine propulsion rate:After building model on the 7th and 21st day,10 rats of random sample in every group were infused to mixture having ⁹⁹mTc-DTPA 0.05mCi/10g and the half solid standard meal with pigment 20 ml/kg. Gastric nuclide remnant rate(%)=(gastric nuclide remnant value/basis value)×100%, intestine propulsion rate(%)=(the distance from pyloric sphincter to the head of pigment(cm)/ the distance from pyloric sphincter to the intestine between ileum and cecum(cm))×100%.6 Pathologic observation on spinal cord tissue:On the 1 st and 7th day after building model,three rats of random sample in normal and model groups were executed, chosen about 1.0cm long tissue in spinal cord regarded T12 as the center,with 4% poly- formaldehyde fixed for 24 hours,normal paraffin wax embedded.After the slices of 5μm thick were done HE dyeing,configurable variety in spinal cord tissue could be observed.7 Motilin(MTL),Gastrin(GAS),Vasoacfive Intestinal Peptide(VIP),Somatostatin (SST)mensuration:The specimens in blood plasma,serum and tissue were collected and put into centrifuge tubes.Then those specimens were centrifuged at 1500 r/min at 4℃for 15 minutes and chosen clear supernatant liquid to be kept in EP tube for Motilin,gastrin,Vasoacfive Intestinal Peptide and Somatostatin assay according to description in radioimmunoassay kit.8 Nitric oxide synthase(NOS)detection:On the 3rd week after building model,rats were executed,then chosen about 1.0cm long tissue of gastric sinuses and duodenum with 4%poly- formaldehyde fixed,normal paraffin wax embedded and made into slice.Making use of Strept Avidin-Biotin Complex method which belongs to immunochemistry technique,the expression of iNOS protein in gastric and intestinal tissue was examined.At the same time the expression of iNOSmRNA was detected by RT-PCR.9 The MTLR mRNA and SSR mRNA gene expression:The expression of MTLR mRNA and SSR mRNA was detected by reverse transcription-polymerase chain reaction(RT-PCR)method,making multimedia gel imaging analysis system compute the light density value of each sample's PCR outcome.Through computing integral calculus light density value of MTL-R1A mRNA,SSTR₂ mRNAs of the each sample vs the outcome of theβ-actins mRNA PCR,the specific value reflected how much the expression of MTL-R1A mRNA and SSTR₂ mRNAs was quantitatively.Results:1 The result of CBS grade of rats in every group after spinal cords injury at different time:The sport function of spinal nerve in the rats of normal group was normal,and the double hind legs of rats in model group appeared paralysis,while ras's muscle tension being low.At the same time various reflections below injured level died down or disappeared in different degree.On the 7th and 22nd day,CBS grade score of model group showed lower evidently very much,compared with that of normal group.2 Gastric nuclide remnant rate and intestine propulsion rate:After one week's test, namely after building model,compared with normal group,each group of spinal cord injury showed extremely obvious difference in gastric nuclide remnant rate and intestine propulsion rate(P＜0.01);After treatment with electroacupuncture,namely after three weeks' test,gastric nuclide remnant rate in model group was significantly higher than that in normal group(P＜0.01),but the electroacupuncture group being obviously lower than that in model group(P＜0.01);After 3 weeks,intestine propulsion rate in model group compared with that in normal group still obviously reduced(P＜0.01),but this ratio in electroacupuncture group being obviously higher than that in model group(P＜0.01);Gastric nuclide remnant rate and intestine propulsion rate in electroacupuncture group remarkably showed difference before and after treatment(P＜0.01).3 Configurable variety of spinal cord tissue:In normal group the HE dye result of spinal cord was normal after 24 hours' building model;While the model group after 24 hours' injury:Through HE dye it was clear that spinal cord tissue lost normal structure and had obvious tissue breakage,bad dead,empty bubble formation in the injured area and adjacent area.It was so clearly seen that a great deal of dispersive red cell inside gray white and white matter.After one week's hurt,in the injured area and adjacent area it was clearly seen that spinal cord tissue lost normal structure,and liquefy to bad dead.Empty bubble formed and internal bleeding disappeared in gray matter and white matter.In the meantime there was a great deal of phlogistic cell and macrophage endosmosis,arranging confusedly,nerve cell rarely being seen.4 Motilin(MTL),gastrin(GAS),Vasoacfive Intestinal Peptide(VIP),Somatostatin(SST) mensuration:Spinal cord injury caould make the content of MTL in the blood and tissue of rats lower;While electroacupuncture let the content of MTL increase obviously in the blood and tissue of Stomach - antrum and duodenum of rats,whose function was equal to western medicine's.The study demonstrated that the content of GAS in blood and tissue decreased significantly accompanied with the suppression of gastrointestinal motility after spinal cord injury,while electroacupuncture at zusanli point could increase the content of GAS in blood of rats obviously.It suggested that acupuncture might regulatie the content of GAS to influence gastrointestinal motility.The experiment suggested that after spinal cord injury the content of VIP increasing in the blood and tissue of rats;Electroacupunctur at zusanli point could affect the content of VIP in the blood and Stomach - antrum tissue of rats obviously, whose function in duodenal tissue was not obvious.Compared with Mosapride,the electroacupuncture's effect on Stomach- antrum tissue was superior to itS.Spinal cord injury could affect the content of SS in the blood and tissue of rats and made it increase.However the treatment with electroacupuncture could lower the content of SS in both the blood and Stomach-antrum of rats obviously,whose function was equal to western medicine's.5 The effect of electroacupuncture on nitric oxide synthase in rats with spinal cord injury:(1)The iNOS expression in gastric and small intestinal tissue:In normal group,the expression of iNOS was small amount in the gastric mucosa epithelia,but in model group and side point group the expression of iNOS obviously increased compared with normal group(P＜0.01).After treatment,the expression in electroacupuncture group and western medicine group obviously reduced(P＜0.01);Compared with normal group,the expression of iNOS obviously enhanced in the model group and side point group(P＜0.01);Through the treatment,the expression in electroacupunctu -re group was down-regulated obviously,and there was a great deal of difference between electroacupuncture group and model group.The result did not show distinct difference between western medicine and normal group.(2)INOS mRNA in gastric and small intestinal tissue:In gastric and small intestinal tissue,model group existed in tendency to enhance the iNOS mRNA gene expression (P＜0.05);Relative to model group and side point group,iNOS mRNA expression in electroacupuncture group decreased distinctly(P＜0.01).6 The regulating effect of electroacupuncture on the expression of gastric MTL-R1A mRNA and small intestinal SSTR₂ mRNA in rats with spinal cord injury:In the MTL-R1A mRNA,the light density integral calculus ratio of model group obviously lowered,compared with normal group,manifesting very obvious difference(P＜0.01); Electroacupuncture group and western medicine group relative to model group,whose average light density integral calculus specific value all enhanced and showed very obvious difference(P＜0.01).In SSTR₂ mRNA,the average light density specific value of the model group obviously went up,compared with normal group,having very distinct difference(P＜0.01);Electroacupuncture group relative to model group, whose specific value lowered and there was very obvious difference between two groups(P＜0.01).It was considered that the MTL-R1A mRNA expression of the rats with spinal cord injury could be down-regulated as a result;Electroacupuncture at zusanli point could had effect on expression of MTL-R1A mRNA,whose function equaled to Mosapride;SSTR₂ mRNA expression of the rats with spinal cord injury could be up-regulated;After treatment with electroacupuncture at zusanli point,the expression of MTL-R1A mRNA could lower,and the function of electroacupuncture was better than that of Mosapride.Conclusion1 According to WD method,rats model with spinal cord injury was established,and improved CBS grade score in model group had obvious difference relative to that in normal group on the 7th and 21st day after building model.The histology observation matched pathologic variety of clinical spinal cord injury,so that it is confirmed to succeed in the establishment of SD rats model with spinal cord injury.2 There were gastrointestinal motive disorder in rats of spinal cords injury,and main expression existed in sport descending of stomach emptying and intestine propulsion rate in this experiment.The results suggested that electroacupuncture could improve gastrointestinal emptying rate,and promote gastrointestinal sport.Therefore, etectroacupuncture had a promotive effect on gastrointestinal motility.3 Electroacupuncture can regulate gastroenteric hormone disorder in the rats with spinal cord injury:(1)In blood plasma and tissue enhancing the content of MTL might result in gastrointestinal motive disorder after spinal cord injury.The content of MTL mainly influenced gastrointestinal sport after spinal cord injury in manner of endocrine and partial neural secretion.Electroacupuncture could promote gastrointestinal motility by means of increasing the content of MTL in blood and tissue.(2)In blood plasma and stomach tissue the content of GAS descending might be one of the pathogenesis,which brought on gastrointestinal function disorder after spinal cord injury.The result indicated GAS influences gastric and intestinal motility after spinal cord injury not only by endocrine,but also by partial neural secretion. Electroacupuncture at zusanli point might encourage the release of GAS in blood and regulate gastroenteric motility induced by endocrine.(3)The content of VIP going up in blood plasma and tissue might be one of the reasons which caused gastroenteric function disorder after spinal cord injury.The regulating effect of electroacupuncture on the contcent of VIP was not only by endocrine,but also through the content of VIP descending in stomach-antrum.As a result electroacupuncture influenced gastrointestinal motility after spinal cord injury by both blood circulation and partial secretion in stomach-antrum tissue.(4)In blood plasma and tissue the content of SS increasing might be one of pathogenesis which brought on gastrointestinal function disorder after spinal cord injury.In rats treated with electroacupuncture at zusanli point the content of SS decreased obviously.This result suggested that electroacupuncture could inhibit the release of SS and regulate gastroenteric motility after spinal cord injury by means of blood circulation and partial secretion.4 The abnormal gene expression of MTL-R1A and SSTR₂ might participate in the outbreak of gastrointestinal motility disorder after spinal cord injury.The study assessed electroacupuncture could up-regulate significantly MTL-R1A gene expression and down- regulate significantly SSTR₂ gene expression,which made the synthesis of MTL-R1A increase and the synthesis of SSTR₂ decrease.It regulated gastrointestina sport after MTL(or SS)and corresponding receptor have combined as so to promote gastrointestinal motility.Therefore,the occurrence of gastrointestinal motility disorder after spinal cord injury was relative to the level of the expression of brain-gut peptide receptor gene in partial tissue,so this disease was probably was relative to the receptors of gastrointestinal hormone.The results indicated that electroacupuncture at zusanli point promoted gastrointestinal mobility through up-regulating or down- regulating the level of mRNA expression of the brain-gut peptide receptor.The mRNA expression of the brain-gut peptide receptor which could make gastrointestinal smooth muscle cell excited decreased,or the mRNA expression of the brain-gut peptide receptor which could make gastroint- estinal smooth muscle cell restrained increased.Electroacupuncture had effect on gastroint- estinal motility after spinal cord injury in manner of the expression of the receptor and influenced the regulation of its gene.5 The study demonstrated that regulating the level of iNOS and the expression of iNOSmRNA might be one of the reasons of gastrointestinal motive disorder outbreak in rats with spinal cord injury.Under the condition of gastrointestinal motive disorder, electroacupuncture could improve the expression of gene and protein in iNOS,then promoting gastrointestinal motility.