| Objective To investigate the effect of intestinal flora in children with functional constipation on the expression of acid-sensitive ion channel 3 in rats,and to explore the regulatory role of ASIC3 in intestinal dynamicsMethod Collect the first affiliated hospital of anhui medical university in December 2017 to June2018 in Rome Ⅳ diagnostic criteria of FC patients and healthy children feces,healthy children feces mixed each other,children with FC feces mixed each other,and then respectively in sterile mixer,join PBS(stool: PBS = 1:10)after mixing evenly,in turn through the pore size of 2.0,1.0,0.5,and0.25 mm mesh filter,homogenate and fecal bacteria liquid after centrifugal separation and purification.All rats were given a mixture of antibiotic water(200mg/Kg vancomycin,200mg/Kg metronidazole and 200mg/Kg neomycin),and a pseudo-sterile rat model was constructed once every other day for 2 weeks.According to the random number table method,the pseudo-sterile rats were randomly divided into the treatment group and the control group,with 12 in each group.The treatment group was treated with fecal bacteria solution for FC children,and the control group was treated with fecal bacteria solution for healthy children by gavage,once a day for 3 days,and once the next day for 7 times in total.Six rats were taken from each group,and each rat was placed separately in a squirrel cage.Feces were collected every 1h,the number of feces particles was recorded and weighed.Two groups of rats fasting water after 16 h,respectively for activated carbon lavage(1.0 m L / 100 g),accurate timing,20 min after chloral hydrate anesthesia,quickly open the abdominal cavity,isolated mesenteric carefully,shearing top since the pylorus,down to the blind of bowel loops back,will be blind without drawing flat out on the glass,use scale to measure the distance of the activated carbon in the small intestine propulsion,calculate small intestine propulsion rate;After fasting for 16 h,and after light anesthesia with ether,the colored plastic beads with a diameter of about 3mm were pushed into the rat’s distal colon(about 3cm away from the anus)through the anus via a smooth plastic rod.Then the rats were placed in a cage with white paper to record the time from placing to discharging the beads,and the colonic power was measured;Six rats in each group were taken and anesthetized with ether.After the rats were anesthetized with ether,one end of the 8F catheter was connected to the mercury sphygmomanometer with a three-way valve.The AWR score was performed at pressures of 20 mm Hg,40 mm Hg,60 mm Hg and 80 mm Hg,respectively;The rats were placed separately in a cage with white paper,the feces were collected and immediately frozen in liquid nitrogen,and the intestinal microorganisms of the rats and FC children were determined by 16 Sr DNA high-throughput sequencing;Rat intestinal tissues 5cm below the pylorus and colon tissues 2cm above the rectum were stored in liquid nitrogen at 1.5cm each,and the expression of intestinal ASIC3 m RNA and protein was determined by fluorescence quantitative PCR and western-blot.Results Compared to the control group,the small intestine propulsion rate,colon motility and visceral sensitivity of the treated group were significantly decreased compared with those in the control group(P=0.017,P=0.005,P=0.043);The species and quantity of intestinal flora of children with FC and rats implanted with FC faecal bacteria were reduced(P=0.045,P=0.023),and firmicutes and bacteroidetes were the main bacterias;the m RNA and protein levels of ASIC3 in the small intestine were down-regulated significantly(P=0.036,P=0.025);and the m RNA and protein levels of ASIC3 in the colon were down-regulated significantly(P=0.013,P=0.031).Conclusions Children with FC have intestinal flora disorder,and Intestinal flora of FC children may affect intestinal motility by down-regulating the expression of intestinal ASIC3 in rats. |
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