| The study was aimed to screen the effective compound Mongolian medicine inhibiting pathogenic Escherichia coli from bovine, and to study its antibacterial mechanism and effect on the intestinal mucosal barrier in rats infected with pathogenic E. coli and the mechanism of action. This study not only lay a foundation for the development of veterinary Mongolian medicine but also to provide data and theoretical basis for clinical application of veterinary compound Mongolian medicine.This study included five experiments. The purpose of the experiment 1 was to isolate and identify pathogenic E. coli from rectal stool of cow, and to study theirs serotype, virulence genes and drug resistance. The purpose of experiment 2 was to screen the best antibacterial compound Mongolian medicine against pathogenic E. coli O1 (containing eaeA, stx1, hlyA and irp24 kinds of virulence genes) through inhibitory test in vitro, orthogonal design, inhibitory test in vivo and augmenting drug test. The experiment 3 was designed on the basis of the experiment 2, and it was to study antibacterial mechanism and antibacterial spectrum of 3 kinds of antibacterial compound Mongolian medicine against pathogenic E. coli O1. The experiment 4 was selected sixty SD rats divided into six groups, the normal control group, the negative control group, the positive control group, the high dose of antibacterial compound Mongolian medicine Ⅲ, the medium dose of antibacterial compound Mongolian medicine Ⅲ and the low dose of compound Mongolian medicine Ⅲ, respectively. Besides the normal control group, rats in other group received treatment as follows over a period of 7 days with exposure to 80% minimum lethal dose E. coli 01 on day 5:saline solution (negative control), ciprofloxacin (0.13 g/kg; positive control), a antibacterial compound Mongolian medicine at a high (2.16 g/kg), medium (1.08 g/kg) and low (0.54 g/kg) dosage. At the end of the experiment, the samples of blood and tissues was collected for detection of relevant indicators of intestinal mucosal barrier. Thereby, the experiment 4 was conducted to study antibacterial compound Mongolian medicine Ⅲ effects on intestinal mucosal barrier in rats infected with pathogenic E. coli O1 and the mechanism of action. The experiment 5 was using MTT assay to determine the best acting time and concentration of antibacterial compound Mongolian medicine Ⅲ and E. coli O1 acting on macrophage RAW264.7 cells. Then, the macrophage RAW264.7 cells were divided into four group, the normal control group, E. coli 01 infection group, the antibacterial compound Mongolian medicine group and the antibacterial compound Mongolian medicine intervention group and cells were cultivated. After culture, determining cell viability, cytokines, ACP, LDH content of cell culture supernatant by ELISA, ACP and LDH kit, and to explore the effect of antibacterial compound Mongolian medicine on cell viability and immune function of macrophages.The results of this study were showed as follows:Experiment 1:A total of 50 pathogenic E. coli were isolated from 100 cow rectal feces samples. In addition,24 pathogenic E. coli isolates belonged to 10 serotypes, of which O18,0146 and 0152 were the predominant serotypes. The virulence genes of irp2, eaeA, stxl, stx2 and hlyA were detected for 100%,50%,14%,10%, and 8% in the 50 pathogenic E. coli isolates and virulence genes bfpA, ST and LT were negative. Moreover, the 10 serotypes of E. coli isolates were multiple drug resistance, they were all resistant to penicillin, tetracycline, doxycycline, rifampin, cotrimoxazole and amoxicillin. In addition, the study found that pathogenic E. coli 01 caused the fastest death and most serious lesions in mice, and contains eaeA, stx1, hlyA and irp24 kinds of virulence genes that give strong pathogenicity to the strain. Therefore, E. coli 01 was choosen as bacterial strain of this study.Experiment 2:This study screened 3 kinds of antibacterial compound Mongolian medicine against pathogenic E. coli 01. Antibacterial compound Mongolian medicine III consisted of 7 medicines:medlar, rheum officinale, fructus chebulae, etc. Antibacterial compound Mongolian medicine IV consisted of 5 medicines:safflower, rheum officinale, fructus chebulae, etc. Antibacterial compound Mongolian medicine V consisted of 7 medicines:angelicae sinensis, scutellaria baicalensis, rheum officinale, etc.Experiment 3:All three antibacterial compound Mongolian medicine inhibited the growth of pathogenic E. coli 01 and protected mice against E. coli 01. The highest protection rate (50%) was obtained for mice treated with antibacterial compound Mongolian medicine III, as same rate as ciprofloxacin treated mice. When acting on the E. coli 01 cells, all three antibacterial compound Mongolian medicine can changed the surface hydrophobicity of the cell and destroyed the integrity of the cell wall over time, and then destroyed the cell membrane and changed the permeability, and then exosmosed the macromolecules in cells, and inhibited expression of protein of E. coli 01. which eventually inhibiting E. coli 01. In addition, all three antibacterial compound Mongolian medicine had widely antibacterial spectrum.Experiment 4: ① The colony counts of Laclobacillus and Bifidobacteria significantly decreased in negative control group compared with normal control group(P < 0.05). However, the colony counts of Lactobacillus and Bifidobacteria were no difference in rats treated with a medium dose of the antibacterial compound Mongolian medicine(P> 0.05). (2) The ITF content in ileum mucosa significantly decreased in negative control group compared with normal control group(P< 0.05). However, ITF content in ileum mucosa was no difference in rats treated with a medium dose of the antibacterial compound Mongolian medicine (P> 0.05). ③ Compared with normal control group, there were an intense swelling, shorting and shedding with villi, and the villi length, the crypt depth, the V/C value, and the thickness of the muscular layer were decreased, and the structure of tight junctions was destroyed and the desmosomes was abnormal in the negative control group. OCCLUDIN, CLAUD IN-1 and Z0-1 protein content and mRNA gene expression levels in ileum mucosa were significantly decreased in negative control group compared with normal control group (P<0.05). Serum DAO, D-lactic acid and endotoxin levels were significantly increased in negative control group compared with normal control group (P< 0.05). Besides, bacterial translocation in negative control group was occurred. However, the above mentioned circumstances in the medium dose of the antibacterial compound Mongolian medicine Ⅲ, the high dose of the antibacterial compound Mongolian medicine III group and the positive control group were significantly improved and repaired. ④ The contents of sIgA, IL-2, IL-10, IFN-y and TGF-β1 in ileum mucosa significantly decreased in negative control group compared with the normal control group (P<0.05). The contents of IL-1β, IL-6, IL-8 and TNF-a in ileum mucosa significantly increased in negative control group compared with the normal control group (P< 0.05). However, compared with negative control group, the contents of sIgA, IL-2, IL-10, IFN-γ and TGF-β1 in ileum mucosa significantly increased and the contents of IL-1β, IL-6, IL-8 and TNF-a in ileum mucosa significantly decreased in the medium dose of the antibacterial compound Mongolian medicine Ⅲ, the high dose of the antibacterial compound Mongolian medicine Ⅲ group and the positive control group. These results showed that the intestinal mucosal barrier can be destroyed by E. coli O1, But it was repaired by treatment with the antibacterial compound Mongolian medicine Ⅲ, especially with the medium dose of the antibacterial compound Mongolian medicine Ⅲ.Experiment 5:The optimal concentration of the antibacterial compound Mongolian medicine Ⅲ acting on RAW264.7 cells was 10-2 g/mL, the best acting time was 12 h. The half cytotoxic concentration of E. coli 01 acting on RAW264.7 cells was 106 CFU/mL, the acting time was 2 h. In E. coli O1 infection group, all mensurated cytokine levels were significantly increased (P<0.05), and cell viability was significantly reduced (P< 0.01), and extracellular LDH activity was significantly increased (P< 0.01), intracellular contents of LDH and ACP were significantly decreased (P< 0.01). The levels of IL-1β、IL-12、TNF-α、IFN-γ and IL-10 were significantly increased (P<0.05) in the antibacterial compound Mongolian medicine Ⅲ group, but the above cytokine levels were significantly lower than that of E. coli O1 infection group. Compared to E. coli 01 infection group, in the antibacterial compound Mongolian medicine Ⅲ intervention group, all mensurated cytokine levels were significantly decreased (P< 0.05), and cell viability was significantly increased (P<0.01), and extracellular LDH activity was significantly decreased (P<0.01), intracellular contents of LDH and ACP were significantly increased (P< 0.01). These results showed that the macrophage phagocytic function and immune function can be improved by the antibacterial compound Mongolian medicine III, so then the antibacterial compound Mongolian medicine Ⅲ can enhanced the phagocytosis and bactericidal effect to invaded E. coli 01. |
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