Screening Of Chinese Medical Formula For Curing Chicken Colibacillosis And Study Of Its Action Mechanism

Chicken colibacillosis. which is caused by avian pathogenic Escherichia coli (APEC), is one of the most significant and widespread infectious diseases occurring in different varieties and age of chicken. It is responsible for large financial losses for the poultry industry each year due to high morbility and mortality. It is also a deuteropathy of New castle disease. avian influenza and other chronic respiratory disease. Antibacterial agents. and inactivated and subunit vaccines are applied to curing or preventing this disease at present. However. the vaccines and antibacterial agents are often invalid due to the variform serotypes of APEC and complicated pathogenic mechanisms. In addition. because of the unreasonable usage of antibacterial agents for long time. the human food security and public health problem are caused. The traditional Chinese medicine has lots of advantages in curative effect. security. and controlling drug resistance and residue. so it is more important for prevention and treatment of livestock infectious disease. Therefore. in this study, we had screened an effective Chinese medical formula through a serial of experiments in vitro and in vivo, then further observed its curative effect and studied its action mechanism from cellular and molecular perspectives. According to these, we hoped to provide a theoretical basis for further research on this Chinese medical formula and the application in prevention or therapy for chicken colibacillosis.According to pertinent literature, we chose 114 kinds of Chinese materia medica including heat-clearing drugs, dampness-eliminating drugs. blood-regulating drugs and others to examine their antibacterial activity against APEC through bacteriostasis test in vitro.6 kinds of Chinese material medica had good antibacterial activity. such as aloe. andrographitis. garlic. calyx seu fructus physalis. fructus mume and granati cortex. Their inhibition zone were respectively 15.12.12.11.11 and 7 mm. Then, we isolated and purified the antibacterial components of aloe by silica gel column chromatography, gel column chromatography and ODS column chromatography. At last, the antibacterial component was identified as fumaric acid through MS. IR and NMR analysis. Then, we further studied the activity of fumaric acid against Escherichia coli. Staphylococcus aureus, Streptococcus. Salmonella. The MIC was respectively 50,50,780 and 780μg/ml and MBC was respectively 100.100,1560 and 780μg/ml.We had screened 39 Chinese medical formula through chicken model infected by APEC. The results showed that No.4. No.5. No.24 and NO.26 had better effect on decreasing mortality. and their mortality were respectively 30%.40%.26.7%. 26.7%. To confirm the effects of these Chinese medical formula. we carried out 2 repeated tests and found that they had stable effects on chicken infected APEC. the mortality were below 46.7%. And No.26 is the best one which the mortality was 26.7% in 2 repeated tests. Then we defined the usage of the combined drugs of No.26. that is Rhizoma Cyperi 40 g. Andrographis paniculata 30 g. Astragalus membranaceus 30 g. and designated it as "Xiang-Qi-Tang"We had further detected the effects of XQT using chicken model infected by APEC. The results showed that XQT could improve food intake and water consumption of infected chicken. The mortality of APEC group was 50%. the middle dose group and high dose group of XQT were respectively 20%(P<0.05) and 13.3% (P<0.01). and the two XQT groups also could improve weight gain. The blood routine results showed that the WBC of APEC group were significantly higher than that of control group (P<0.01). the WBC of low. middle and high dose group of XQT were significantly lower than that of APEC group (P<0.05 or P<0.01):the number of thrombocyte of APEC were significantly lower than that of control group (P<0.05). and the number of high dose group of XQT were higher than that of APEC group (P<0.05):XQT also could protect red blood cell. Then. XQT could decrease the typical pathological change of infected chicken, including decreasing incidence of pericarditis and perihepatitis and improving organ indexes of heart, liver, spleen and kidney. Through ELISA method, we detected the levels of TNF-a. IL-1 and sEPCR in serum of infected chicken, the results showed that XQT could decrease these inflammation mediator. These results indicated that XQT had better effect on chicken infected by APEC. which might be through anti-inflammation and anti-coagulation to play its role.Microvascular endothelial cells play an important role in pathogenic process of APEC. Therefore, in this study, we detected the effects of XQT and its active components on coagulation. through the inflammation model of MVECs induced by LPS. MVECs was treated with XQT and its active components for 3h. then challenged with 1μg/mL LPS. the cells were incubated at 37℃in a cell incubator for 18h. The supernatants were collected and analyzed the contents of PAI-1 and TF by ELISA kits. The results showed that as compared with LPS control group, the PAI-1 contents were significantly decreased in the low. middle and high dose group of XQT andα-Cyperone. in the middle and high dose group of AstragalosideⅣ, and in the low dose Andrographolide;the TF contents were significantly decreased in the low. middle and high dose group of XQT. in the middle and high dose group ofα-Cyperone. and in the high dose group of AstragalosideⅣand Andrographolide. It suggested that XQT and its active components could inhibite the secretion of PAI-1 and TF, which could decrease coagulation.We analyzed effect of XQT and its active components on mRNA levels of PAI-1, TF and KLF2 using semi-quantitive RT-PCR. The results showed that, after MVECs challenged with LPS for 6h. as compared with LPS control group. the levels of PAI-1 mRNA significantly decreased in XQT group. while the levels did not decrease inα-Cyperone. AstragalosideⅣand Andrographolide group; the levels of TF mRNA significantly decreased in XQT andα-Cyperone group. while the levels did not decrease in AstragalosideⅣand Andrographolide group:the levels of KLF2 mRNA significantly decreased in XQT.α-Cyperone. AstragalosideⅣ. Andrographolide group. These results suggested that XQT and its active components could inhibit the levels of mRNA of PAI-1 and TF induced bv LPS. and regulated the levels of mRNA of KLF2 which was the regulator of PAI-1 and TF. We analyzed effect of XQT and its active components on expression of ICAM-1, IκB-αand NF-κB p65 using ELISA and western blot. The results showed that as compared with LPS control group. the levels of mRNA TNF-a significantly decreased in low, middle and high dose group of XQT and a-Cyperone. in middle and high dose group of Astragaloside IV and Andrographolide. The expression of ICAM-1 protein significantly decreased in XQT. a-Cyperone. Astragaloside IV and Andrographolide. These results suggested that XQT and its active components had better anti-inflammation. In addition. XQT and its active components did not increase the expression of IκB-α. while they could decrease the expression of NF-κB p65. It suggested that the anti-inflammation action of XQT and its active components was mediated by decreasing the expression of NF-κB p65.Based on all the information above, we obtained an effective Chinese medical formula. XQT. curing chicken colibacillosis through in vitro and in vivo screening. XQT could improve water consumption. food intake, mortality, weight gain. blood routine. typical pathological change. organ indexes and inflammation mediator. And the effect of XQT might be performed through regulating the coagulation mediated by KLF2 and inflammation mediated by NF-κB.