Effects Of Stachyose And Fruit Enzymes On The Constipation Model And Intestinal Flora Of SD Rats And Its Mechanism

Objective:To explore the regulatory effect and mechanism of stachyose,a common prebiotic,and fruit enzymes?apple enzymes,mulberryenzymes?on loperamide-induced constipation model and intestinal flora in SD rats.Methods:Forty-eight 6-week-old male and SPF SD rats were randomly divided into 6 groups?n=8 in each group?:normal group?N group?,model group?M group?,positive control group?AT group?,stachyose intervention group?ST group?,stachyose-apple enzyme mixed intervention group?SAT group?and stachyose-mulberry enzyme mixed intervention group?SMT group?.The normal group?group N?was intragastrically infused with 0.9%normal saline throughout the experiment.The other 5 groups of SD rats were given loperamide 20 mg/?kg.d?intragastrically once a day for 8 days,and the effect of the model was evaluated according to the water content of feces and the time of the first black stool.After the completion of the model,the rats were intervened for 8consecutive days.In the process of intervention,the rats in group N and group M were given intragastric administration of normal saline.The AT group was treated by intragastric administration of Senna leaf extract with crude drug concentration of 5.0×10^-2g/ml.The ST group was given stachyose dose of 3.0g/d/person?for 50kg body weight?,which was converted to 12 mg/ml stachyose saline solution in rat body weight for intragastric administration.The enzyme components of SAT group and SMT group were composed of fruit enzyme solution and normal saline at 3:1,and the mixed stachyose concentration was 12 mg/ml.The intestinal motility ratio?%?of rats was measured after intervention.After intestinal tissue treatment,HE staining and immunohistochemical treatment were performed to observe and analyze the effect of intervention on intestinal tissue structure of SD rat model of loperamide constipation.16s rDNA PCR-DGGE was used to analyze the difference of intestinal flora in SD rats before and after intervention.The relative contents of Bifidobacterium,Lactobacillus,Bacteroidesand Firmicutesin the intestinal flora of SD rats were detected by real-time q PCR.The relative expression of AQP3 and AQP8 in colonic epithelium of SD rats was detected by RT-q PCR.Results:After intervention,the time of first black stool in AT group,SAT group,ST group and SMT group was significantly lower than that in M group?P<0.05?.After intervention,the fecal water content in the AT group and SAT group was significantly higher than that in the M group without significant.As for the M group,the fecal water content in the ST group and SMT group was significantly higher than that in the M group?P<0.05?.After intervention,the intestinal propulsion ratio in AT group,SAT group,ST group and SMT group was significantly higher than that in M group.The colonic performance of rats in AT group,SAT group,ST group and SMT group was significantly better than that in M group.Histologically,after intervention,the colonic mucosal epithelium of rats tended to recover,the arrangement of glands was regular,and the mucosal layer returned to filling and swelling.The colonic muscular layer and submucosa of rats in SAT group,ST group and SMT group were significantly thicker than those in M group.The expression of AQP3 and AQP8 protein in colonic epithelium was detected by immunohistochemical method.The immunohistochemical score of AQP3 in SAT group,SMT group and ST group was higher than that in M group,but there was no significant difference between groups.The AQP8 immunohistochemical score of colon in M group and ST group was significantly higher than that in AT group?P<0.05?.16s rDNA PCR-DGGE analysis showed that there were differences in intestinal microflora between group N and other model-made groups of SD rats after modeling.After intervention,stachyose combined with fruit enzyme promoted the intestinal flora of constipation model rats to be close to group N,and the approaching degree of group N in AT group and SAT group was closer than that in ST group and SMT group.The results of 16s rDNA PCR-DGGE preponderant strip cutting sequence analysis showed that there were bands 1 and 2 with difference in band position and brightness in group N,and the results showed that band 1 might be Lactobacillus gasseri ATCC 33323=JCM1131,and the homology is 98.15%.Stripe 2 may be Lactobacillus agilis strain La3,with 88.75%homology.In group M,bands 3,4,5,6 and 9 had differences in band position and brightness.The results showed that band 3 might be Anoxybacillusamylolyticus strain DSM 15939 chromosome,homology is 94.12%.Band4 may be Lactobacillus animalis KCTC 3501=DSM 20602 NODE?24,homology is97.40%.Band 5 may be Paraclostridiumbifermentans ATCC 638 gcd ATCC638.contig.17,homology of 97.08%.Band 6 may be Thermincolaferriacetica strain Z-0001Tfer?ctg45,homology is 82.22%.Band 9 may be Bacillus oryziterrae strain ZYK contig6,with 80.12%homology.The No.10 band in the SAT group has a difference in band position and brightness,and the results show that the band may be Pectinophilus ATCC 43243 Scfld?02?1,homology is 97.01%.Bands 7 and 8 in SMT group had differences in band position and brightness.The results showed that band 7 could be homologous to Hungatellahathewayi WAL-18680 supercont1.6,with a homology of84.89%.Stripe 8 can be Anaerostipescaccae DSM 14662 Scfld?03?6,with a homology of 89.86%.The intestinal flora showed that The ratio of Bifidobacterium was significantly higher than that in M group,N group,SAT group and ST group?P<0.05?.The ratio of Lactobacillus was significantly higher than that in M group,N group and SMT group?P<0.05?.The ratio of Bacteroides was significantly higher than that in M group,N group,SAT group and ST group?P<0.05?.The ratio of Bacteroidetes/Firmicutes was significantly higher in SAT group and ST group than that in M group?P<0.05?.However,the ratio of Bacteroidetes/Firmicutes decreased significantly in N group and SMT group?P<0.05?.The expression of AQP3 and AQP8 in colon tissue was detected by q RT-PCR.The expression level of AQP8 protein in N group,SMT group and ST group was significantly lower than that in M group?P<0.05?.Conclusion:After intragastric administration of loperamide 20mg/?kg/d?for 8 days,the constipation model of SD rats could be established.During the intervention for 8 days,the constipation model of SD rats could be maintained and could not be completely restored to normal state.Constipation caused a significant decrease in the relative content of Bifidobacterium,Lactobacillus and Bacteroides in the intestinal flora of SD rats,while the Bacteroidetes/Firmicutes in the intestinal flora in the constipation group was significantly higher than that in the N group.When the human dose of stachyose was used,stachyose combined with apple enzyme or mulberry enzyme could improve the constipation symptoms of SD rat constipation model.Among them,mulberry enzyme combined with stachyose not only had a better effect on improving the symptoms of constipation model in SD rats,but also significantly increased the content of Lactobacillus in intestinal flora,improved intestinal flora and decreased the expression level of AQP8 protein in colonic epithelium.