Effects Of Genistein On Intestinal Microbiota Of Ovalbumin Induced Allergic Diarrheal Mice

In recent years, food allergy has an increasing tendency. Many reports have showed that food allergy was related to the change of intestinal microbiota. However, which microorganisms change in food allergy? What are the effects of microorganisms on food allergy? There is so far limited information available. Since it has been difficult to avoid source of food allergen, prebiotics has been introduced as a new method to prevent and cure food allergy. Genistein was a kind of phytoestrogen which could have many benefits to humans. To investigate the relationship between allergic diarrhea and intestinal microorgnisms, and whether genistein can attenuate allergic diarrhea disease and its possible microbial mechanism, this study was conducted to investigate the effects of genistein on fecal microbiota, colonic microbiota and colonic mucosal microbiota of OVA induced diarrheal mice using PCR/DGGE and real-time PCR techniques.In the first section,32female BALB/c mice were randomly allotted to four groups: negative group (N), positive group (P), low genistein group (L) and high genistein group (H). Each group had eight mice, fed with diet without soybean in group N and group P, while in group L and group H fed with diet without soybean adding genistein10mg/kg and100mg/kg respectively, injected intraperitoneally and induced by intragastric administration with OVA. OVA induced diarrheal model was established successfully. ADFI of four groups was significantly decreased on2day、15day and33day. The real genistein intake was1.22in low genistein group and12.19(mg/kg BW) in high genistein group after correction. Diarrheal mice emerged on32day and the number of diarrheal mice increased from32day to38day. Average body weight of four groups tended to decrease between32day and38day. The status of diarrheal mice on38day:N group mice did not have diarrhea, while those in group P and group H had heavy diarrhea, and group L mice had alleviative diarrhea.In the second section, PCR/DGGE was used to investigate the effect of genistein on feceal microbiota of OVA induced diarrheal mice on different days. DGGE analysis revealed that no apparent difference in microbiota was observed between groups in feces of28day. One band appeared in DGGE in feces of38day only in low genistein group while not in other groups. The diversity index of diarrheal feces decreased significantly as compared with the normal feces, the newly appeared special bands of group H had high similarity with Clostridium which may have something with diarrhea. Trail DGGE analysis revealed that, there was no difference in fecal bacterial community of different days in the same group, the diversity index of diarrheal feces decreased significantly, band6emerged but band7disappeared in group H. In conclusion, OVA sensitization had no effect on fecal bacterial community, but diarrheal feces had lower diversity index. Genistein was likely to enrich certain bacterial species which may be related to diarrhea.In the third section, DGGE and real-time PCR were used to investigate the effect of genistein on colonic microbiota of OVA induced diarrheal mice on38day. DGGE analysis revealed that gut microbial diversity of group P mice was significantly lower than those of N group. Within the same group, the similarity index of mice was high, while low similarity was observed between groups (P group and L group). QPCR showed that compared with those in group N, mice in group P, L and H had higher number of16S rRNA gene copies of total bacteria in the colon, significantly lower copies of Bifidobacterium, relatively stable copies of E.coli and Clostridium cluster Ⅳgroup. Compared with group N and group L, copies of Lactobacillus in group P decreased significantly. Compared with group L, copies of Bacteroidetes in group H increased significantly. Conclusion:The bacterial diversity in diarrheal mice decreased, with copies of Lactobacillus and Bifidobacterium decreased. Mice sensitized and induced by OVA had higher copies of total bacteria and lower copies of Bifidobacterium. Low level of genistein (10mg/kg) alleviated the diarrhea and increased copies of Lactobacillus comparing with group P and group H, suggesting that the alleviation of diarrhea by genistein may be related to increase the copies of Lactobacillus.In the last section, to investigate the effect of genistein on colonic mucosal microbiota of OVA induced diarrheal mice on38day, DGGE and real-time PCR were used to analyze the microbiota. DGGE analysis revealed that there was difference in bands and the similarity index in different groups. QPCR showed that it had stable copies of total bacteria and Bacteroidetes, but compared with those in group N, mice in group P, L and H had higher percentage of Bacteroidetes in total bacteria, group P had an increasing tendency and group H had a significant increase. Compared with those in group N, mice in group P, L and H had higher copies of Lactobacillus, group L had a significant increase, group P had a increasing tendency. Compared with those in group N and in group L, mice in group P and H had lower copies of E coli, it had a significant difference between group P and group L. Conclusion:Comparing with group N and group L, mice in group P and group H had higher percentage of Bacteroidetes in total bacteria and lower copies of E.coli. Mice sensitized and induced by OVA had higher copies of Lactobacillus. Group L alleviated the diarrhea and increased copies of Lactobacillus comparing with group N, suggesting that the alleviation of diarrhea by genistein may be related to increase the copies of Lactobacillus.