Preventive Effects And Mechanisms Of Baicalin On Chicken Colibacillosis

Chicken Colibacillosis is a local or systemic infectious disease in chickens caused by avian pathogenic Escherichia coli(APEC),which is the number one bacterial disease threatening the poultry industry and causes enormous economic losses to poultry farming every year.It is generally believed that the occurrence of chicken colibacillosis begins with a respiratory infection by APEC.APEC adheres and colonizes in the air sac and lung epithelium and induces local inflammatory response,and then break through the air-blood barrier into the blood circulation,and finally results in systemic symptoms such as pericarditis and pericarditis.Therefore,inhibiting the lung inflammatory responses induced by APEC and mataining the integrity of air-blood barrier are the key means to protect against chicken colibacillosis.Due to the traditional theory “lung and the large intestine being interior-exteriorly related” believes that the,the treatment means of “regulating the intestine function to treat lung dieases and regulating the lung function to treat intestines dieases” were recorded as early in Treatise on Typhoid and Miscellaneous Diseases.Furthermore,according to modern conception “gut-lung axis”,the interaction between lungs and intestines has been paid more and the intestinal flora and its metabolites has been proved to be an important link between lungs and intestines.Meanwhile,there is increasing evidences reported that Chinese herbal medicines can exert their pharmacological effects by interacting with the intestinal flora.Furthermore,with the arrival of the era of “reduced antibiotics” and “limited antibiotics”,Chinese herbal medicine has become the first developed medicine for the prevention and treatment of livestock diseases based on its advantages such as wide source,low residue and not leading to the emergence of drug-resistant strains.Scutellaria baicalensis,a traditional Chinese medicine in China,is the dried root of the labiatae family plant Scutellaria baicalensis Georgi.It has a bitter taste,cold in nature,and has the effects of clearing heat and dampness,clearing lungs and purging fire,clearing heat and detoxification.Baicalin,the major flavonoid extracted from the Scutellaria baicalensis,has a variety of pharmacological effects,such as antibacterial,antiviral,antitumor,anti-inflammatory,antioxidant,and modulating the intestinal flora effects.Therefore,we first explored the protective effect of baicalin against chicken colibacillosis by the model of chicken type ? pneumocytes and chickens infected by APEC.Secondly,we investigated the role of gut microbiota played in chicken colibacillosis after depleted the gut microbiota by broad-spectrum antibiotics.Subsequently,we examined the alteration of gut microbiota and its metabolites for further analyzing the regulatory effect of baicalin on gut microbiota and its metabolites,and then screening the specific bacteria and metabolites associated with the anti-colibacillosis effect.This study aimed to reveal the protective mechanism of baicalin against chicken colibacillosis,so as to provide research ideas and theoretical guidance for the prevention and treatment of chicken colibacillosis and the develop of related drugs.First,we infected chicken type ? pneumocytes by APEC,and then examined the concentration of baicalin that had no effect on the growth of APEC and had no toxicity on type II pneumocytes of baicalin by MIC and MTT assay was 12.5?g/mL?25 ?g/mL ? 50 ?g/mL;Then,we detected the effects of different concentrations of baicalin on the release of LDH,bacterial adhesions,and the gene expression of inflammatory cytokines in chicken type II pneumocytes after APEC infection.The results showed that baicalin reduced the release of LDH and inhibited the adhesion of APEC to cells and the gene expression of proinflammatory cytokines TNF-?,IL-1?,and increased expression of anti-inflammatory cytokine IL-4.In vivo,the chickens were pretreated with 50 mg/kg,100 mg/kg,and 200 mg/kg of baicalin for a week,and then the model of chicken colibacillosis was established by intratracheal inoculation with APEC.Next,we tested the chicken mortality,lung histopathological changes,bacterial loads,and gene expression of inflammatory cytokines and tight junction proteins in lung tissues to evaluate the preventive effect of baicalin on chicken colibacillosis.The results showed that baicalin pretreatment significantly reduced the chicken mortality,inhibited lung tissue injury,suppressed the gene expression of TNF-?,IL-1? and IL-6 and increased gene expression of tight junction proteins ZO-1,occludin,and Claudin-3 in the lung induced by APEC.These results suggest that baicalin inhibited lung inflammatory responses induced by APEC and maintained the integrity of the air-blood barrier,thus preventing chicken colibacillosis.Next,to explore the role of gut microbiota in chicken colibacillosis,we depleted the gut microbiota by adding broad-spectrum antibiotics(Abx)to the drinking water for two weeks,and then established the model of chicken colibacillosis by intratracheal instillation of APEC.The organ indexes and pathological changes,as well as the expression of inflammatory cytokines and tight junction proteins in lung tissues were detected.The results showed that the the pathological damages in the lung,heart,liver,and intestine were significantly serious,and the production and gene expression of TNF-?,IL-1?,and IL-6 were significantly increased in Abx+APEC group comparing with APEC group.Examination of the permeability of the air-blood barrier revealed that gut microbiota depletion resulted in significantly increasing proteins concentration in bronchoalvellar lavage fluid(BALF),and significantly decreasing the gene expression of tight junction proteins Claudin-3,occludin,and ZO-1 in lung tissue in Abx+APEC group.These results indicated that gut microbiota depletion aggravated organ damages and increased the permeability of air and blood barrier induced by APEC.Subsequently,to further explore whether the protective effects of baicalin against APEC-induced chicken colibacillosis was connected with gut microbiota,the control chickens and microbiota-depleted chickens were pretreated with baicalin for week and then infected by APEC.We detected the organ indexes and pathological changes,as well as bacterial loads,production and gene expression of inflammatory cytokines and gene expression of tight junction proteins in lung tissues.The results showed that the effects of baicalin inhibiting the pathological damages of organs and inflammatory cytokines releases and expression as well as reducing the bacterial loads,BALF protein and restored of expression of tight junctional protein in lung tissues were significantly weakened after gut microbiota depletion.These results suggested that gut microbiota played an important role in baicalin against AEC-induced colibacillosis in chicken.In order to further explore whether baicalin played a protective role in chicken colibacillosis by regulating gut microbiota,we examined the alteration of gut microbiota in control(CON),APEC,baicalin treatment alone(BAI)and baicalin pretreatment and infection with APEC(BAI+APEC)groups.The results showed that the abundance and diversity of gut microbiota in APEC group were significantly increased comparing with control group,and abundance of Actinobacteria and Ruminococcaceae?UCG-014,Ruminococcaceae?unclassified,Clostridiales?vadin BB60?group?Unclassified and ruminiclostridium?5 were obviously increased while the abundance of Proteobacteria,lachnospiraceae?unclassified,blautia,Escherichia Shigella,and pygmaiobacter were significantly decreased.while the altered abundance and structure of gut flora caused by APEC infection were significantly restored in the baicalin pretreatment group.However,baicalin pretreatment significantly restored the changes in the abundance and structure of gut microflora induced by APEC infection.Further LEf Se analysis showed that baicalin treatment mainly increased the abundance of SCFAs-producing bacteria in the gut,such as g?Blautia,f?Lachnospiraceae and g?Intestinimonas.In addition,to exclude the effect of baicalin on lung microflora,we further analyzed the lung microflora in the CON and BAI group.The data indicated that baicalin had no effect the structure of lung microflora.These results suggested that baicalin regulated restored the dysbiosis of intestinal microbiota induced by APEC and increased the abundance of SCFAs-producing bacteria.Finally,to investigate whether baicalin played a protective role in chicken colibacillosis by promoting the production of gut microbiota metabolites SCFAs,we examined the concentrations of SCFAs in the intestinal and BALF in the CON group and BAI group by targeted metabolomics.Our results showed that baicalin treatment significantly increased the concentration of the acetic acid,propionic acid and butyric acid in intestinal,and the concentration of acetic acid and butyric acid and the acetic acid receptor FFAR2 expression in lungs were significantly increased.Therefore,we hypothesized that baicalin may play an anti-inflammatory role and maintain the function of air-blood barrier by promoting the production of microflora metabolites SCFAs,especially acetic acid,and then the increased acetic acid migrates to the lung to activate its receptor FFAR2.To verify the above hypothesis,we added acetate in the drinking water of chickens and then establish the model of chicken colibacillosis by intratracheal instillation of APEC.And then we explored the preventive effect of acetate on chicken colibacillosis.The results showed that acetate supplementation in drinking water significantly inhibited the pathological damages of lung tissues,the production and gene expression of inflammatory cytokines TNF-?,IL-1? and IL-6 caused by APEC infection,as well as increased the gene expression of tight junction proteins Claudin-3,Occludin and ZO-1 and acetate receptor FFAR2 in lung.These results suggested that baicalin may protect against chicken colibacillosis by promoting the production of intestinal SCFAs,especially acetic acid,which migrated to the lung and activates its receptor FFAR2 to resist APEC infection and prevent chicken colibacillosis.In summary,this study demonstrated the preventive effects of baicalin on chicken colibacillosis.The specific mechanisms may be as follows: baicalin restored APEC-induced dysbiosis of gut microbiota and increased the abundance of SCFAs-producing bacteria,thereby promoting the production of intestinal SCFAs,especially acetic acid.And then the high concentration of SCFAs(especially acetic acid)migrated from the intestinal tract to the lungs and activated FFAR2 to protect against chicken colibacillosis by inhibiting pulmonary inflammatory injury caused by APEC and maintaining the integrity of air-blood barrier.This study not only provided a new research idea and experimental basis for the prevention and treatment of chicken colibacillosis in clinical application,but also provided a new direction for the research of natural compounds.It also provided theoretical basis for detoxification of the basic theory of Chinese medicine of “lung and the large intestine being interior-exteriorly related”.