Influence Of Baicalin On The Mice In The Aspects Of The Changes Of Intestinal Flora

ObjectiveIntestinal tract is an open cavity, there were varieties of microbes living in it, mainly bacteria, commonly referred to as intestinal flora. Under the ordinary status, the intestinal flora was in a balance with the human body and its external environments, and played an important role in physical fitness. But there has had a lot of reports referred that overdose of antibiotics would cause the alteration of intestinal flora. Lead to many diseases. According to the Chinese traditional medical theory, large dosages and/or long term usage of bitter and cold traditional Chinese medicine would injure the spleen-qi and waste the healthy energy, bitter and cold traditional Chinese medicine are widely used in clinical. with the anti-inflammatory, bactericidal effect, clearing and detoxifying and other effects. But there are not any reports about whether abusage of bitter cold traditional Chinese medicines can cause the similar katogene as antibiotics. This study was undertaken to try to investigate the influence of Baicalin on the mice in the aspects of the changes of intestinal flora, use traditional bacterial culture methods and PCR-DGGE method to observe the diversity of intestinal bacteria, colony structure and the intestinal tissue damages and so on. Investigate the law of baicalin on intestinal flora. Look forword to provide experimental reference for the rational use of bitter and cold traditional Chinese medicine.Methods1.Constract the control groups of enteric dysbacteriosis mice, use the cillimycin, constract the study groups, imbue with different doses of baicalin, and the blank group, imbue with the saline water. Observe the appearance, behavior, diet and so on of the mice in each groups. mices were sacrificed by breaking neck after 14 days, remove the cecal content, extract the DNA of bacteria and conduct Gene amplification with the PCR instrument. Then conduct DGGE. analysis the fingerprint, bacterial diversity and abundance of species of the results.2. Constract the control groups of enteric dysbacteriosis mice, use cillimycin, constract the study groups, imbue with different doses of baicalin, constract the normal control group, imbue with saline water. All groups are divided into different time:three-day,five-day,nine-day,fourteen-day. Observe the appearance, behavior, diet and so on of the mice in each groups. Then mice are sampled from the eyeball blood, under ether anesthesia, detection Lymphoid cells changes. Then mice are sacrificed by breaking neck. Remove the cecal content, examine the amount of Lactobacillus, Bifidobacterium, Enterococcus, Escherichia colibacillus in the cecal content of each mouse.Following the procedures of weighing,clarifixatio,diluting,dripping,spreading,culturing and counting. Then analyzing the data in eath group with SPSS statistical software. Make the pathological section of ileum, then observe the intestinal tissue conditions with light microscopy, and compare the differences between groups.Results1.PCR-DGGE fingerprinting technique analysis of bacteria:Low-dose baicalin group showed highest Shannon-Wiener index, species richness in the four groups; Cillimycin group showed lowest Shannon-Wiener index, species richness in the four groups; Bacterial species of normal saline group, cillimycin group and low-dose baicalin group showed high degree of intragroup similarity and high intergroup diversity. middle-dose baicalin group showed high degree of intragroup and intergroup diversity.2. Plate culture and counting statistics showed(1)Changes of E.coliThree-day:the number of E. coli in high-dose baicalin group is increasing compared with the normal group, the difference is obvious (P<0.05);Five-day:the number of E. coli in low-dose baicalin group is increasing compared with normal group, the difference is significant(P<0.01);Nine-day:the number of E. coli in cillimycin-group, low-dose baicalin, high-dose baicalin group are all increasing(P<0.05,0.01,0.01 respectively)compared with normal group;Fourteen-day:the number of E. coli in low-dose baicalin, middle-dose baicalin, high-dose baicalin group, cillimycin-group are all increasing compared with normal group, the difference is significant(P<0.01);But the increased number of low-dose and middle-dose baicalin group is another kind of E. coli colony, this colony is more common in low-dose baicalin group than middle-dose baicalin group. The colony of high-dose baicalin group is the same as normal group and cillimycin group.With the experimental time increasing, cillimycin group:The number of E.coli is increasing, the results of parewise comparison(refer to appendix 4):the number of fourteen-day is increasing significantly compare with the first three time(P<0.05);Low-dose baicalin:the number of E. coli is fluctuating, and has a growing trend in the end, the number of three-day is smallest. compared with the three time later(P<0.05);Middle-dose baicalin group:the number of E.coli is fluctuating significantly. the number of fourteen-day is the most. compared with the first three time(P<0.05);High-dose baicalin group and normal group:the number of E. coli is smooth, there is no statistical significance between each time.(2)Changes of EnterococciThree-day:the number of enterococci in middle-dose, high-dose baicalin group is decreasing, compared with normal group, the difference is significant (P<0.01). Five-day:the number of enterococci in middle-dose baicalin is decreasing significantly compared with normal group(P<0.01). Nine-day:the number of enterococci in cillimycin-group is increasing compared with normal group, the difference is obvious(P<0.05);Fourteen-day:the number of enterococci in cillimycin-group, high-dose baicalin group is increasing compared with normal group(P<0.01,0.05 respectively);the number of middle-dose baicalin is decreasing compared with normal group, the difference is significant(P<0.01)With the experimental time increasing, the number of enterococci in cillimycin-group is increasing,the number of the three time later are increasing significantly compared with three-day(P<0.05)(refer to appendix 4);the number of enterococci in high-dose baicalin is increasing,the fourteen-day, nine-day is increasing obvious compared with three-day, five-day(P<0.05);the number of enterococci in middle-dose baicalin is fluctuating significantly. the number of nine-day is the most, compared with another three time, the difference is obvious(P<0.05);the changes of normal group and low-dose baicalin group have no statistical significance.(3)The changes of BifidobacteriumThree-day, five-day:the number of bifidobacterium in each group has no statistical significance. Nine-day:the number of bifidobacterium in cillimycin group, high-dose baicalin group is decreasing, compared with normal group, there is statistical significance(P<0.05,0.01);Fourteen-day:the number of bifidobacterium in cillimycin group, high-dose baicalin group is decreasing significantly, compared with normal group(P<0.01).With the experimental time increasing, the number of bifidobacterium in cillimycin-group is decreasing gradually, the number of fourteen-day is the least compared with the first three time, there is statistical significance (P<0.05) (refer to appendix 4);the number of bifidobacterium in high-dose baicalin group is decreasing, the three-time later compared with three-day, there is statistical significance(P<0.05);the number of bifidobacterium in middle-dose baicalin group is fluctuating significantly. and has the trend of increasing. the number of fourteen-day is the most, compared with the first three time (P<0.05); the number of normal group is smooth; the number of low-dose baicalin group has the trend of increasing. there is no statistical significance between each time.(4)Changes of LactobacillusThree-day:the number of lactobacillus in each group has no statistical significance. Nine-day:the number of lactobacills in cillimycin group, high-dose baicalin is decreasing, compared with normal group, there is statistical significance(P<0.05,0.01);Nine-day:the number of lactobacills in cillimycin group is decreasing, compared with normal group, there is statistical significance (P<0.05);Fourteen-day:the number of lactobacills in Cillimyin group, high-dose baicalin group is decreasing significantly, compared with normal group(P<0.01,0.05).With experimental time increasing, the number of lactobacills in cillimycin-group is decreasing gradually, the number of fourteen-day is the least compared with the three-day, there is statistical significance (P<0.05)(refer to appendix 4);the number of low-dose baicalin group has the trend of increasing. the later three time is increasing obviously compared with three-day(P<0.05);the number of high-dose baicalin group has the trend of decreasing and fluctuating, the number of three-day is the most, compared with the later three time(P<0.05);the number of normal group is smooth;the number of low-dose baicalin group has the trend of increasing, there is no statistical significance between each time.3. Pathological section of ileum under light microscopy showed:normal group:the structure of ileum are intact; With the experimental time increasing, Low-dose baicalin group:the structure of ileum are normal, epithelium are integrity, glandular are neat, villus are rich;middle-dose baicalin group:ileum mucosa are thinner, epithelial part of loss,villus are fewer and shorter, from the ninth day;high-dose baicalin group and cillimycin group:ileum mucosa were thinner gradually, interstitium were mild congestive, epithelium were dropping, the number of goblet cells were fewer, villus were dropping.4. Lymphoid cells statistics showedThree-day, Five-day:the number of lymphoid cells in each group is no statistical significance. Nine-day:the number of lymphoid cells in cillimycin group, high-dose baicalin group is dereasing, compared with normal group, there is statistical significance (P<0.01,0.05);Fourteen-day:the number of lymphoid cells in cillimycin group, high-dose baicalin group is decreasing significantly, compared with normal group(P<0.01).With the experimental time increasing, cillimycin group:the number of lymphoid cells is decreasing gradually, compared to normal group with a statistically significance(P<0.01). Low-dose and middle-dose baicalin group:the number of lymphoid cells is increasing gradually;There is all no statistical significance between each group.high-dose baicalin group:the number of lymphoid cells has the trend of decreasing, nine-day, fourteen-day compared with five-day, three-day, the decreasing of lymphoid cells is obvious(P<0.05)(refer to appendix 4).Conclusions1. Low-dose baicalin, the structure of ileum were intact, showed highest Shannon-Wiener index, species richness in four groups;structure of flora was stable, the number of beneficial bacteria were increasing, and the number of enterococcus(a kind of opportunistic pathogen) was decreasing;at the same time, it induced the new flora. This situation suggested that, Low-dose baicalin maybe have the influence of regulating the balance of intestinal flora, and could promote a more stable colony structure.2. The mice in the high-dose baicalin group, suffered from the dysbacteria of intestinal flora and intestinal injury, similar with the cillimycin group;the middle-dose baicalin group also suffered from the dysbacteria of intestinal flora and intestinal injury in the later periods, and the structure of flora was unstable. This situation suggested:large dosages and/or long term usage of baicalin have the similar injury effect as antibiotics.