| Objective:Wistar adult rats were seleted as the research object of this subject by colorectal mechanical stimulation to replicate the IBS-D animal model.This experiment proved the effectiveness of the suppressing Mu component of Tongxieyaofang.The expression of BDNF mRNA in hippocampus and colon was quantitatively analyzed by real-time fluorescence.And Using 16 SrRNA technology find disciplinarian about intestinal flora of IBS-D animal model,the suppressing Mu component of Tongxieyaofang regulate intestinal microflora to alleviate visceral hypersensitivity of IBS-D related to BDNF-TrkB signaling pathway.This study will provide new ideas and methods for the treatment of IBS-D.Methods:Model group:adult male Wistar rats were accepted colorectal dilatation(pressure 80mmHg)for 14 days,given adequate food and water.Fourty rats were randomly selected ten rats as blank control group,and the rest were model rats.After one week,Abdominal withdrawal reflex(AWR)and colorectal pathological examination were used to determine whether the model was successful or not.Successful rats were randomly divided into three groups: model group(n = 10),Baishaofangfeng group(n = 10),Chenpibaizhu group(n = 10).The next day,they were fed with the durg.They were fed once a day,for 14 days.During the experiment,each rat was observed the general condition,body weight and stool condition.The weight of each rat was weighed before and after the establishment of the model and the treatment.The stools of each group were recorded before and after treatment(Bristol classification of stools).The abdominal wall retraction of each rat was detected after the establishment of the model and after the treatment.Fresh stool specimens were collected after treatment,and 8 cm of distal colon and rectum and hippocampus tissue were removed.The hippocampus and colon tissues of five rat in each group were used to dotest.Total RNA was extracted from the samples,then RNA are synthesized from cDNA by reverse transcriptase,the target fragments were finally amplified and synthesized by using DNA as template.The expression level of BDNAmRNA was obtained by total RNA extraction,reverse transcription reaction and real-time fluorescence quantitative polymerase chain reaction.The fecal specimens collected from 5 rats in each group were analyzed by 16 S rRNA technology,Illumina platform sequencing and bioinformatics analysis technology.The changes of intestinal flora structure in each group were compared by data accusation,OTU clustering and distribution taxonomy.Results:(1)General observation of rats: blank group rats: rats in general good condition,smooth hair,color,eating normal,sensitive to external stimuli.The feces were normal and there was no diarrhea.The rats in the model group showed increased vigilance on the second day of modeling and were too sensitive to external stimuli;on the fourth day,the rats in the model group showed poor spirits,disordered hair,reduced food intake and loose stools;on the second day after administration,the rats in the Baishaofangfeng group improved their spirits,increased their food intake and basically formed their stools;on the seventh day after administration,the rats in the Chenpibaizhu group showed spirits.After the model was established,the stool of some rats was not formed.(2)Compared with the blank group,the weight gain of the model group was less than that of the blank group(P < 0.05).After the treatment,the body weight of rats in Baishaofangfeng group and Chenpibaizhu tangerine group was higher than that in model group(P < 0.05),but the effect of Baishaofangfeng group on body weight was obvious.(3)After modeling,the Bristol Stool Scale score of the model group was significantly lower than that of the blank group(P < 0.05),and the Bristol Stool Scale score of the model group was higher than that of the blank group(P < 0.05).After treatment,the Bristol Stool Scale score of the Baishaofangfeng group and the Chenpibaizhu was lower than that of the model group(P < 0.05).(4)Visceral pain threshold of rats: Compared with the blank group,the pain threshold of the model group was significantly lower(p < 0.05).After treatment,the visceral pain threshold of the rats in Baishaofangfeng group was higher than that of the rats after modeling(P < 0.05).The visceral pain threshold of the rats in the orange peel and Chenpibaizhu group was not significantly improved after treatment(P < 0.05).There was no significant difference(P < 0.05).(5)Pathological examination of rat specimens: There was no significant inflammatory cell infiltration and interstitial edema in the colonic epithelium of rats in each group,which was consistent with the pathological changes of IBS-D.(6)The expression of BDNFmRNA in hippocampus and colon tissue of rats: In colon tissue,the expression of BDNFmRNA in model group was significantly higher than that in blank group(P<0.01).Compared with model group,the expression of BDNFmRNA in colon of rats in BaishaoFangfeng group was significantly lower(P <0.05).There was no significant difference in the expression of BDNFmRNA in colon of rats in Chenpibaizhu group(P>0.05).In hippocampus,the expression of BDNFmRNA in model group was significantly lower than that in blank group(P<0.01).Compared with model group,the expression of BDNFmRNA in colon tissue of rats in Baishaofangfeng group increased significantly(P < 0.05);the expression of BDNFmRNA in hippocampus of rats in Chenpibaizhu group increased significantly(P < 0.05).(7)The effect of intestinal flora in rats:The flora diversity of IBS-D rats decreased.Compared with the blank group,the intestinal flora of IBS-D rats decreased,the thick-walled flora increased and the Proteus flora increased.Baishaofangfeng group and Chenpibaizhu group could increase Bacteroides and decrease the abundance of Thickwall bacterium and Proteus.Conclusion:The suppressing Mu component of Tongxie Yaofang can improve the visceral hypersensitivity of IBS-D model rats.Its mechanism may be through improving the intestinal flora structure and adjusting the differential expression of BDNF in brain and intestine. |
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