| Purpose:We take patients with functional constipation as the object of clinical research,compare the effect of electro-acupuncture with mosapride on functional constipation,verify the clinical effection on electro-acupuncture and analyse its curative characteristics,provide objective evidence for optimizing the therapeutic schedule of functional constipation.In the experimental study,rats with constipation induced by compound diphenoxylate tablets were used as models.In order to find the intrinsic mechanism of treating constipation with electro-acupuncture and provide new theoretical basis on finding potential therapeutic targets for constipation,we observe the effects of electro-acupuncture,western medicine,combinating with the acupuncture and western medicine on functional constipation.We observe changes of content of intestinal nervous system(ENS)and related neurotransmitters(SP,NO,VIP,SS)and markers(GFAP,ox-42),inflammatory factors(IL-1? and IL-6)in colonic tissues and blood in rats.And the effects on inflammatory factors IL-1? and IL-6 in the serum and colon tissues of constipated rats.Material and method: Paper One: Observation on clinical effect of electro-acupuncture Tianshu and Shangjuxu point on functional constipation 72 patients who met the inclusion criteria for functional constipation were divided into electro-acupuncture group(treatment group)and Western medicine group(control group),36 cases in each group.In the electro-acupuncture group,needles were used to acupuncture bilateral bilateral Tianshu and Shangjuxu pionts.The bilateral bilateral Tianshu points were connected to SDZ-II Huayu brand electro-acupuncture treatment instrument,using continuous wave,frequency 3Hz,current intensity should be well tolerated by patients,20 min each time,once a day,5 days a week at week1-2,3 days a week at week3-4,totally 16 days.In the western medicine group,subjects were required to take mosapride citrate tablets orally,5 mg/time,3 times a day for 4 weeks.CSBM,SBM,BSFS,CCS and PAC-QOL of constipated patients were recorded respectively at 1 week before treatment and 2,4 and 8 weeks after treatment.Observe the clinical effect of electroacupuncture and western medicine on functional constipation.Paper Two: Experimental study of the effect of electro-acupuncture Tianshu and Shangjuxu point on functional constipation Part One: Prepare for animal model with functional constipation(slow transit)in rats and observe its general state 1.Method of Grouping and Modeling Fifty SPF rats were randomly divided into 5 groups(blank group,model group,electro-acupuncture group,western medicine group,combination group)according to the body weight,10 in each group,male and female in half.In addition to the blank group,rats in other groups were intragastrically administered with Compound Diphenoxylate Tablets at 10mg/kg/d.The capacity of intragastric administration was 1m L/100 g,once a day for 14 days.And then the animal models of functional(slow transit)constipation were established.2.Observation index Body weight,fecal traits,hair,activity,and fecal water content were observed before and after modeling in each model group and blank control group.Part Two: Treatment of constipation model rats and the measurement of rate of intestinal carbon transition ? Electro-acupuncture group: 0.25mm×25mm acupuncture needles were used to acupuncture bilateral bilateral Tianshu and Shangjuxu pionts,with a depth of 5-7mm.The bilateral bilateral Tianshu points were connected to SDZ-II Huayu brand electroacupuncture treatment instrument,using continuous wave,frequency 2Hz,current 1m A,15 min each time,once a day,5 days a week,totally 14 days.? Western medicine treatment group: Gavage with suspension made from Mosapride.Calculate the amount of gavage according to the weight of rats(1.35mg/kg/d),the volume of intragastric administration was 1m L/100 g,totally 14 days.? Electro-acupuncture+Western medicine group: Treatment combined with electro-acupuncture and western medicine.On day 15,all rats were fasted for 24 hours,and intragastrically administered with with suspension composed of 5% carbon powder and 0.9% sodium chloride at 1m L/100 g.Abdominal anesthesia with 7% chloral hydrate(0.5ml/100g).take the tissues from pylorus to the end of rectum and measure the rate of intestinal carbon transition.Part Three: Sampling of serum and colonic tissue of rats.After successful intraperitoneal injection of anesthesia,abdominal aorta blood was collected for 5ml and the serum was centrifuged for 10 minutes at 4? and 3500r/min,and stored at-80?.After blood collection,the tissues needed were cut out quickly.1.The colonic parts of each rats were taken and immediately washed with 0.9% sodium chloride solution to remove the blood and impurities,then quickly wrapped in well-marked tinfoil paper and put into liquid nitrogen tank.Ultimately stored at-80 °C for western blot and ELISA.2.Sampling after perfusion with 4% paraformaldehyde,the sampling tissues were fixed with 4% paraformaldehyde for 3-5 days,and Sedimentated with 30% sucrose for 24 hours.Embed the tissues with OCT and make frozen slices with freezing microtome(-25 ?)for immunohistochemical staining.Part Four: Detect the expression of Neurotransmitters and Inflammatory Factor in Serum and Colonic Tissues of Rats.The levels of Neurotransmitters SP,NO,VIP,SS and inflammatory factors IL-1?and IL-6 in the intestinal nervous system(ENS)were detected by ELISA.Part Five: Detect the expression of intestinal glial cell markers GFAP and OX-42 in rat colon.The expression levels of glial cell markers GFAP and OX-42 were detected by Western blot and immunofluorescence.Results: 1.Observation on clinical effect of electro-acupuncture Tianshu and Shangjuxu point on functional constipation.There were no significant differences in sex,age,weight,frequency of complete spontaneous bowel movement(CSBM)and spontaneous bowel movement(SBM)every week,Bristol stool form scale(BSFS),Cleveland Clinic Score(CCS)and Patient Assessment of Constipation Quality of Life(PAC-QOL)between the two groups before treatment(P >0.05).Frequency of CSBM and SBM every week,BSFS,CCS and PAC-QOL are all improved in both group after treatment and the difference was statistically significant(P < 0.05).2.Preparation of animal model with functional constipation(slow transit)in rats and measurement of rate of intestinal carbon transition In this study,we established animal models of constipation in rats with compound diphenoxylate tablets successfully.Compared with the blank group,rats in the model group showed the following characteristics: 1)Less increasing in body weight,2)Dull hair and physical inactivity,3)feces become cracked,thinner and shorter.The fecal moisture percentage and rate of intestinal carbon transition in model group were lower than the blank group(P <0.05).3.Effects of electro-acupuncture on the expression of intestinal neurotransmitters and inflammatory factor in colonic tissue Excitatory transmitter: 3.1 SP: The content of SP in the model group was significantly lower than the blank group(P < 0.001).Compared with the model group,the content of SP in the combined group increased significantly(P <0.001),the electro-acupuncture group and the western medicine group also showed a trend of increase,but there was no statistical difference.3.2 VIP: The content of VIP in the model group was significantly lower than the blank group(P < 0.001).Compared with the model group,the content of SP in the electro-acupuncture group and combined group increased significantly(P<0.001).No statistical difference was found between the western medicine group and the model group.Inhibitory transmitter: 3.3 NO: The content of NO in the model group was significantly higher than the blank group(P < 0.001).Compared with the model group,the content of NO in both the electro-acupuncture group(P<0.05)and the combination group(P<0.001)decreased significantly,the western medicine group also showed a trend of decrease,but no statistical difference was found.3.4 SS: The content of SS in the model group was significantly higher than the blank group(P<0.001).Compared with the model group,the content of SS in the electro-acupuncture group(P<0.001),the western medicine group(P<0.01)and the combination group(P<0.001)all decreased significantly.Inflammatory factor: 3.5 IL-1?: The content of IL-1? in the model group was significantly higher than the blank group(P<0.001).Compared with the model group,the content of IL-1? in both the electro-acupuncture group and the combination group decreased significantly(P<0.01),the western medicine group also showed a trend of decrease,but no statistical difference was found.3.6 IL-6: The content of IL-6 in the model group was significantly higher than the blank group(P<0.001).Compared with the model group,the content of IL-6 in both the electro-acupuncture group and the combination group decreased significantly(P<0.01),the western medicine group also showed a trend of decrease,but no statistical difference was found.4.Effect of electro-acupuncture on the expression of intestinal neurotransmitters and inflammatory factors in blood.Excitatory transmitter: 4.1 SP: The content of SP in the model group was significantly lower than the blank group(P<0.001).Compared with the model group,the content of SP in the combined group increased significantly(P<0.05),the electro-acupuncture group and the western medicine group also showed a trend of increase,but there was no statistical difference.4.2 VIP: The content of VIP in the model group was significantly lower than the blank group(P<0.001).Compared with the model group,the content of SP in both the electro-acupuncture group and combined group increased significantly(P<0.001).There was no significant difference between western medicine group and model group.Inhibitory transmitter: 4.3 NO: The content of NO in the model group was significantly higher than the blank group(P < 0.001).Compared with the model group,the content of NO in both the electro-acupuncture group(P<0.05)and the combination group(P<0.001)decreased significantly,the western medicine group also showed a trend of decrease,but there was no statistical difference.4.4 SS: The content of SS in the model group was significantly higher than the blank group(P<0.001).Compared with the model group,the content of SS in the electro-acupuncture group(P<0.05)and the combination group decreased significantly(P<0.001),the western medicine group also showed a trend of decrease,but there was no statistical difference. Inflammatory factor: 4.5 IL-1?: The content of IL-1? in the model group was significantly higher than the blank group(P<0.001).Compared with the model group,the content of IL-1? in both the electro-acupuncture group and the combination group decreased significantly(P<0.01),the western medicine group also showed a trend of decrease,but no statistical difference was found.4.6 IL-6: The content of IL-6 in the model group was significantly higher than the blank group(P<0.001).Compared with the model group,the content of IL-6 in the electro-acupuncture group(P<0.01),the western medicine group(P<0.05)and the combination group(P<0.01)all decreased significantly.5.Effects of electro-acupuncture on intestinal glial cells in rats with functional constipation(slow transit).The expression of GFAP and OX-42 in colonic tissues were detected by Western blot and Immunofluorescence.Compared with the blank group,the expression of GFAP and OX-42 in the model group increased significantly(P <0.05).,which suggests the activation and dysfunction of intestinal glial cells.The expression of GFAP and OX-42 in the electro-acupuncture group,the western medicine group and the combination group were significantly lower than the model group(P <0.05).Conclusion: 1.The clinical efficacy of electroacupuncture at Tianshu point combined with Juxue point in the treatment of functional constipation is good.Its effect is comparable to that of western medicine mosapride,and it has a fast onset and has certain persistence.There are no side effects of western medicine.It is worthy of clinical promotion.2.The administration of compound diphenoxylate tablets can successfully establish a functional(slow transmission type)constipation rat model,which is in line with the clinical characteristics of constipation patients and can be used in animal experiments.3.It is confirmed by animal experiments that the occurrence of functional(slow transmission)constipation may be related to the decrease of excitatory neurotransmitter SP(substance P),VIP content and the increase of inhibitory neurotransmitters(SS,NO)in rats.It may be related to the increased content of inflammatory factors(IL-1?,IL-6)in rats;it may be related to the activation expression of intestinal glial cells(microglia,astrocytes)in colon tissue.4.Electroacupuncture treatment can improve the stool characteristics of constipation rats and improve the intestinal propellate rate of constipation rats.5.Electroacupuncture treatment of constipation may be related to up-regulation of excitatory neurotransmitter SP,VIP expression,down-regulation of inhibitory neurotransmitters SS,NO and inflammatory factors IL-1? and IL-6 expression;may also reduce constipation The activation expression of intestinal glial cells(GFAP,OX-42 protein)is correlated in rat colon tissue. |
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