Anti-infective Effect Of Schizandrin On Avian Pathogenic Escherichia Coli (APEC)

The virulence factors and biofilm formation of avian pathogenic Escherichia coli(APEC)are important factors for the increase of drug resistance in APEC,and the abuse of antibiotics also aggravates the drug resistance of APEC to some extent.The effective components of traditional Chinese medicine can inhibit the virulence factors and biofilm formation of bacteria without affecting the growth of bacteria.This method can effectively reduce the survival pressure of bacteria and is not easy to produce drug resistance.Therefore,in this study,firstly,the anti-APEC activity of schizandrin was studied in vitro to determine its minimum inhibitory concentration(MIC)and antibacterial mechanism.On this basis,the concentration of drug that did not inhibit the growth of bacteria was selected to detect the effect of schizandrin on the formation of APEC biofilm.Secondly,the inflammatory injury of chicken typeⅡpneumocytes infected by APEC and the lung lesion caused by APEC infection were established to explore whether schizandrin can reduce the inflammatory injury caused by APEC infection and protect the host.The research contents are as follows:First of all,the MIC of schizandrin to the test strain APEC-O78 was 1600μg/mL,by agar dilution method,which confirmed the effective bacteriostatic effect of schizandrin.The effect of schizandrin on the growth curve of APEC-O78 was determined.It was found that schizandrin affected the logarithmic growth phase of APEC-O78 and inhibited the division of APEC-O78.The extracellular contents of alkaline phosphatase(AKP)and extracellular nucleic acid were selected as the index of whether the cell wall or cell membrane was damaged.The results showed that the content of AKP and extracellular nucleic acid increased significantly after APEC-O78was treated with schizandrin at the concentration of 1/2MIC and MIC.It was observed by scanning electron microscope that the smooth structure of the cell surface was destroyed,the bacteria was distorted and even lysed after treatment with schizandrin.The above results show that schizandrin can delay the growth of APEC-O78,shorten its growth cycle,destroy the integrity of cell wall,change the permeability of cell membrane and exert antibacterial activity.Based on the results of the first chapter,schizandrin(50μg/mL,100μg/mL and200μg/mL)with no effect on the growth of APEC was selected,and the effects of schizandrin on the swimming and swarmming motility of APEC were observed by using LB semi-solid medium with different concentrations of agar.The results showed that schizandrin could inhibit the motility of APEC-O78.At the same time,the biofilm formation was quantitatively detected by crystal violet staining and the microstructure of biofilm was observed by scanning electron microscope.It was found that schizandrin could reduce the adhesion of bacteria to the carrier surface and inhibit the formation of APEC-O78 biofilm.The content of signal molecule AI-2 in APEC quorum sensing system was detected,and it was found that schizandrin reduced the AI-2 production of APEC-O78.The effect of schizandrin on the expression of biofilm related genes was detected by RT-PCR.The results showed that schizandrin significantly decreased the expression levels of biofilm related genes such as fliC,csgA,csgB,fimA,rpos,fyuA,iucD,H-NS,luxS,pfs,lsrK and lsrB.These results suggest that schizandrin can resist APEC infection and reduce bacterial drug resistance by inhibiting the formation of biofilm.In the second chapter,the results have shown that schizandrin can significantly down-regulate the mRNA level of adhesion-related genes.In this chapter,through the establishment of the model of chicken typeⅡpneumocytes infected by APEC-O78,we found that schizandrin could reduce the adhesion number of APEC to chicken typeⅡpneumocytes,which was consistent with the result of gene expression.At the same time,the degree of cell injury was reflected by measuring the content of LDH.The results showed that schizandrin could significantly reduce the release of LDH from chicken typeⅡpneumocytes induced by APEC-O78,which may be achieved by anti-APEC adhesion.The effects of schizandrin on the secretion of TNF-α,IL-1β,IL-6 and IL-8 in chicken typeⅡpneumocytes induced by APEC-O78 were determined by ELISA and RT-PCR.The results showed that schizandrin significantly decreased the production of inflammatory cytokines and the expression of mRNA in chicken typeⅡpneumocytes induced by APEC-O78,which confirmed the anti-inflammatory effect of schizandrin.The anti-inflammatory mechanism of schizandrin was studied by western blot.The results showed that schizandrin significantly decreased the phosphorylation levels of MAPK p38,JNK,NF-κB p65and IκB,suggesting that schizandrin exertes its anti-inflammatory effect through NF-κB and MAPK signal pathway.The purpose of this chapter was to explore the molecular mechanism of schizandrin in alleviating lung lesion by estalishing the model of lung lesion in chickens infected with APEC.The chicken lung lesion model was successfully induced by intraperitoneal injection of APEC-O78 and 1.8×10~9 CFU.The bacterial load of organs was examined 24 hours later,and the results showed that schizandrin significantly reduced the bacterial load of heart,liver,spleen and lung.The lung tissue was collected to examine the histopathological changes.The results showed that schizandrin significantly reduced the pathological lesion of lung tissue in APEC infected chickens in a dose-dependent manner.The wet/dry ratio(W/D)of lung tissue was measured to reflect the degree of lung edema.The results showed that schizandrin reduced the degree of lung edema induced by APEC.The content of MDA in lung tissue was determined.The results showed that schizandrin could significantly reduce the production of MDA and improve the antioxidant status of lung tissue.The secretion level of inflammatory cytokines was measured by ELISA method and the anti-inflammatory mechanism was studied.The results showed that schizandrin significantly decreased the phosphorylation levels of MAPK p38,ERK,JNK,NF-κB p65 and IκB in lung tissue,and inhibited the production of inflammatory factors such as TNF-α,IL-1β,IL-6 and IL-8 through NF-κB and MAPK signal pathway.To sum up,this study shows that schizandrin has good bacteriostatic activity against APEC at high drug concentration(>1600μg/mL).Without inhibiting the growth of APEC,schizandrin can inhibit the formation of APEC biofilm and the inflammatory reaction caused by APEC,which proves that schizandrin can reduce the pressure of bacterial survival and exert its anti-infective effect by inhibiting the physiological process of APEC.Our study will not only provide theoretical data for the prevention and treatment of avian pathogenic Escherichia coli,but also provide new ideas for the study of active components of traditional Chinese medicine,which will have important reference significance.