Quantitative Analysis Of Intestinal Flora Status In Different Large Intestinal Segments Of SD Rats With Hepatic Injury By Real-Time PCR

Recent studies showed that intestinal microecology and liver tie up closely not in anatomical structure but also in functional status, they can influenced each other. It can exacerbate the injury of the liver and subsequently induce various complications. Recent experiments and clinic trials are mainly concerned with the effect of hepatic failure, liver cirrhosis on the intestinal flora and based on the specimen examination of rectum feces, and yield out the decrease of beneficial bacteria like Bifidobacterium and Lactobacillus whereas the increase of harmful bacteria like Enterobacteriaceae. Datas of the intestinal flora changes related to chronic hepatic injury are rare, and the colony number and distribution of intestinal flora haven't been reported yet. Thus the present experiment is designed as follows:Materials and MethodsPart one: Quantitative Analysis of Intestinal Flora Status in Different Large Intestinal Segments of SD Rats By Real-Time PCRAnimals and Sample:SD rats, male vs. female 1:1, weight 150±10g, were enrolled in this study. The fresh faeces were analyzed as samples.Methods:1. Quantitative analysis of intestine flora by traditional method.2. Standard plasmid DNAs containing different interest fragments according to the aim of investigation were constituted and transformed into DH5αcells(E.coli)3. The real-time PCR was established with SYBR GreenⅠ, and we quantified the targeted faecal bacteria of all subjects.4. All results were analyzed by SPSS(Version 10.0).Results The results analyzed by traditional method showed that intestine flora in the Normal Group reached the peak in almost equivalent amount in the following three segments: the caecum, the colon and the rectum, which consistents with the results by other researches. The results analyzed by real-time PCR showed intestine flora were equivalent amount in the following three segments: the caecum, the colon and the rectum. The results were relative to that from traditional culture method.Part two: Quantitative Analysis of Intestinal Flora in Rats with Acute Hepatic Injury By Real-Time PCR and Effect of"GANSHENGYUAN"or Spirulina on itExperiment design: SD mice were divided into five group(Normal group, Model group,"GANLIXIN"(GLX) group,"GANSHENGYUAN"(GSY-1) group and Spirulina group), each group contained 10 rats. Normal group received 2m1 sterile water by gavage from 5th day before injection of 25% CCl4(5ml/kg). While GLX group received GLX (15mg/kg·d ), GSY-1 group received GSY-1(45mg/kg·d), Spirulina group received Spirulina (500mg/kg·d ). Gavage lasts until the sacrifice of the animals.Specimen collection was collected on the 2th day after injection of 25% CCl4(5ml/kg) (n=10). The rat was sacrificed and liver enzyme, plasma endotoxin, tumour necrosis factorα(TNF-α) and liver histological changes were measured. Six mice of each group were random selected for investigating bacterial counts in different intestinal segments (caecum, colon, and rectum).ResultModel group showed a decrease of Bifidobacterium counts and a increase of Enterobacteriaceae and Enterococcus in intestine fragments: caecum, colon, and rectum. This results analyzed by traditional method were similar to those from real-time PCR. The levels of the plasma endotoxin and TNF-αincreased.Pretreatment with GLX, GSY-1 and Spirulina can both lowed the levels of the plasma endotoxin and TNF-α48h after injection of 25% CCl4(5ml/kg). In GSY-1 group and Spirulina group showed a increase in Bifidobacterium counts and a decrease in Enterobacteriaceae and Enterococcus; GLX, GSY-1 and Spirulina can lowed the levels of the plasma endotoxin and TNF-α24h after acute liver injury, which were associated with decreased liver function such as ALT, AST and DBIL.Part three: Quantitative Analysis of Intestinal Flora in Rats with Chronic Hepatic Injury By Real-Time PCR and Effect of GSY-1 or Spirulina on itExperiment design: SD mice were divided into five group(Normal group, Model group, GLX group, GSY-1 group and Spirulina group), each group contain 10 mice. Four groups except Normal group were subcutaneously injected with CCl4, diluted with peanut oil in advance, at a dosage 0.2ml/kg. The concentration of CCl4 ascended 5%: 5%, 10%, 15%, 20%, 25%, 30%. CCl4 injection lasted 3 months, totally 12 times. Normal group received 2m1 sterile water by gavage from 5th day before injection of CCl4. While GLX group received GLX (15mg/kg·d ), GSY-1 group received GSY-1(45mg/kg·d), Spirulina group received Spirulina (500mg/kg·d ). Gavage lasts until the sacrificed of the animals.Specimen collection was collected on the 2th day after injection of 25% CCl4(5ml/kg) (n=10). The rat were sacrificed and liver enzyme, plasma endotoxin, tumour necrosis factorα(TNF-α) and liver histology were investigated. 6 mice were random selected for investigating bacterial counts in different intestine segments (caecum, colon, and rectum).Model group showed a decrease of Bifidobacterium counts and a increase of Enterobacteriaceae and Enterococcus in intestine fragments: caecum, colon, and rectum. This results analyzed by traditional method were similar to that from real-time PCR. The levels of the plasma endotoxin and TNF-αwere increased, t the levels of PLD,TGF-β1 and HA were also increased.Pretreatment with GLX, GSY-1 and Spirulina can both lowed the levels of the plasma endotoxin, TNF-α, PLD, TGF-β1 and HA. In GSY-1 group and Spirulina group showed a increase in Bifidobacterium counts and a decrease in Enterobacteriaceae and Enterococcus; GLX, GSY-1 and Spirulina can lowed the levels of the plasma endotoxin and TNF-α24h after chronic liver injury.ResultsAccording to quantitative analysis by real-time PCR, the results of intestinal flora were consistent with the results by traditional culture.The above results also showed that disturbance of intestinal flora was observed in rats with acute or chronic liver injury. The elevation of plasma endotoxin and TNF-αwas also found. Supplemented with prebiotics, probiotics or symbiotics, such as GSY-1 and Spirulina, can decrease the levels of plasma endotoxin liver injury through improving intestinal microflora. Real-Time PCR is an effective method to analyze the intestinal flora.