| 1.ObjiectiveIn our previous study,we have shown that Phellodendron Barkeri has good efficacy in surgical abdominal infections.In this study,based on the previous study,we observed the inhibitory effect of the compound Phellodendron Bifidum on Escherichia coli and biofilm;also observed its effect on infected wounds and its molecular mechanism.2.Methods2.1 In vitro experiments on the inhibition of Escherichia coli and its biofilm by Phellodendron Bark extract(1)Single colonies of Escherichia coli were picked in 3 m L of MH and incubated at37℃ for 12 hours with 150 rpm shaking to determine the OD600 value of 1.The minimum inhibitory concentration of the compound cypress liquid coating was determined by the two-fold dilution method.(2)Single colony of Escherichia coli was picked in 3 m L of LB,incubated at 37℃and 150 rpm for 12 hours and the OD600 value was determined to be 1.The bacterial solution and the drug concentration of 0.5 m L/m L,1 m L/m L and 1.5 m L/m L were added to four 100 m L of LB liquid medium,incubated at 37℃ and 150 rpm with shaking,and the OD600 of each group was measured every 2 h and analysed.The OD600 of each group was measured and analyzed.(3)The drug concentrations of 0μL/m L,125μL/m L,250μL/m L,375μL/m L and500μL/m L were incubated at 37℃ for 12 h and then diluted in a 10-fold serial gradient.100μL of 10-5 and 10-6 gradient concentrations were selected and spread on LB solid medium plates and incubated at 37℃ to observe The number of colonies was observed.(4)The concentrations of 0μL/m L,100μL/m L,200μL/m L,300μL/m L,400μL/m L and500μL/m L were set and added to 48-well plates and sterilized tubes respectively,making a total of 1000μL per well of 48-well plates and 5m L per tube,and incubated for 24 h at 37°C to observe the crystalline violet The biofilm was stained.The amount of biofilm formation was measured by an enzyme marker OD492 after lysis in acetic acid.(5)Cell crawls of 3 mm in diameter were placed in 48-well plates,and a mixture of drug concentrations of 0 μL/m L,100 μL/m L,200 μL/m L,300 μL/m L,400 μL/m L and 500 μL/m L was added and incubated at 37°C for 24 h.The cell crawls were fixed,dehydrated,dried,mucilaginous and gold sprayed and then observed by SEM.Biofilm.2.2 Molecular mechanism of inhibition of Escherichia coli biofilm by compound cypress liquid coating A single colony of Escherichia coli plate was selected and inoculated in 5 m L of LB liquid medium,incubated at 37℃ for 12 hours with 150 rpm shaking,and the OD600 value was set at 1,and then diluted 1:1000 in fresh LB liquid medium.3 m L of bacterial solution was added to 2 sterilized centrifuge tubes,in which no drug was added to the control group and 400 μL/m L to the experimental group.The bacteria were incubated at 37℃ for 12 hours and then centrifuged at 8000 rpm for 2 min and resuspended twice in sterile PBS buffer.The effect of the compound Phellodendron cypress solution on the expression level of genes related to biofilm formation in E.coli was verified by transcriptome sequencing and library construction using RT-qPCR.2.3 Animal experiments of compound Phellodendron Bifidum liquid application on Escherichia coli biofilm formation after infected wounds A mouse trauma model of E.coli biofilm infection was established by scalding the back of mice and injecting 0.5 × 108 CFU of bacterial solution(100 μL).The Blank group(Blank),the Model group(Model),the compound cypress solution application group(HB)and the iodophor group(DF)were set up.The DF group was for mice infected with the biofilm and cleaned with iodophor disinfectant.On day 3,7,11 and14,three mice were executed in each group,and the healing rate and number of days of healing were observed.3.Results3.1 In vitro experiments on the inhibition of Escherichia coli MPEC6 and its biofilm by Phellodendron Bifidum(1)The minimum inhibitory concentration(MIC)of E.coli MPEC6 was measured at500 μl/m L by the twofold dilution method.(2)The growth curve of the bacteria showed that the positive control and 1/2 times the drug concentration groups without the addition of Phellodendron cypress solution showed rapid growth in the delayed and logarithmic phases,and slow growth in the later phases.The bacteria grew significantly slower at a drug concentration of 500μl/m L drug and the growth gradually decreased as the drug concentration increased.(3)CFU experiments showed that when the drug concentration was 125 μl/m L,there was no difference compared with the positive control group without drug(P>0.05);when the drug concentration was 250 μl/m L,there was a significant difference compared with the control group(P<0.01);when the drug concentration was 500μl/m L,the growth of MPEC6 was inhibited.(4)After the 48-well plate and glass test tube biofilms were intervened by the drug,when the drug concentration of compound cypress liquid coating reached 300 μl/m L,there was a significant difference(P < 0.01)compared with the control,and as the drug concentration increased,the stronger the inhibitory effect of compound cypress liquid coating on the biofilm of Escherichia coli MPEC6.(4)Scanning electron microscopy could be used to observe the disruption of the biofilm of E.coli by the compound cypress solution.3.2 The transcript levels of genes related to the biofilm of Escherichia coli were affected by the compound cypress liquid coating(1)Using transcriptome sequencing of 4402 genes,184 gene transcripts were defined as DEGs,of which treatment with cypress solution resulted in 74 DEGs being up-regulated(p ≤ 0.05)and 110 DEGs being down-regulated,and these differentially expressed m RNAs were significantly different in terms of biological processes,cellular components and molecular functions.The enrichment analysis revealed that DEGs were mainly involved in the pathways of microbial metabolism,flagellar assembly,biofilm formation and bacterial secretion system under adverse conditions.(2).RT-qPCR verified that compound cypress liquid coating upregulated the transcription of mar A and omp F through biofilm formation,resulting in a 1.9-fold and3.2-fold increase in the transcript levels of mar A and omp F,respectively,compared to the control group.3.3 Intervention study of compound Phellodendron liquid coating on Escherichia coli after biofilm trauma infection(1)Changes in body weight of mice in each group There was no statistical difference in the body weight of mice in each group before and 3,7,11 and 14 days after moulding(P>0.05).(2)Bacterial count on the trauma surface On the 7th day after moulding,three mice were taken from each group,and the results were obtained after diluting the bacterial solution in a gradient manner: no colony formation was seen in the Blank group;355.67±50.02 in the Model group;104.67±8.32 in the HB group;151±14.17 in the DF group;the bacteria were found in the HB,DF and Model groups.There were significant differences between HB group,DF group and Model group(P<0.01),and there were differences between HB group and DF group(P<0.05),and the efficacy of compound cypress liquid coating was better than that of iodophor.(3)Wound healing in mice a.Comparison of wound healing rate.At day 7,the healing rate was(28.72±3.33)% in the Blank group,(6.56±0.76)% in the Model group,(30.15±2.45)% in the HB group and(22.89±2.83)% in the DF group.there was a statistical difference between the Model group and the Blank group(P<0.05),the Blank group and the HB group There was no difference between the Blank and DF groups and the HB and DF groups(P > 0.05);at day 11,the healing rate was(24.95 ± 6.27)% in the Blank group,(52.05 ± 0.72)% in the Model group,(68.51 ± 0.87)% in the HB group and(46.40 ± 2.02)% in the DF group.The Blank and DF groups healed better than the Model group,with significant differences between the two groups(P < 0.05),the HB group healed better than the Model,Blank and DF groups,with differences between the groups(P < 0.05),and there were no differences between the Blank and DF groups(P > 0.05);at day 14,the Blank group was(77.78 ± At day 14,the healing rate was(77.78±2.47)% in the Blank group,(66.65±0.52)% in the Model group,(93.73±0.81)% in the HB group and(81.48±3.54)% in the DF group.There was no difference between the Blank and DF groups(P>0.05).b.Comparison of wound healing time of mice in each group.The mice in the Blank group took(18.33±0.33)days to heal their wounds,the mice in the Model group took(27±0.57)days to heal their wounds,the mice in the HB group took(19±0.57)days to heal and the DF group took(23.33±0.88)days to heal.the Model group took longer to heal than the Blank,HB and DF groups and there was a statistical difference(P< 0.05);the DF group healed slower and took longer to heal than the HB and Blank groups,with a difference(P<0.05),while there was no difference between the HB and Blank groups(P>0.05).(4)HE staining results On day 3 after modelling,inflammatory exudate with interstitial oedema and scattered lymphocytic infiltrates were seen in all groups.On day 7,the inflammatory exudate was still visible in all groups,with a small amount of collagen fibres in the Blank group and more lymphocytic infiltrates in the Model group.On day 11,the inflammatory cell infiltration was significantly less in the Model,HB and DF groups than before,but the inflammatory cell infiltration was still evident in the Model group,with a significant proliferation of new epidermal cells and a large number of collagen fibres visible in the HB group and less so in the DF group.On day 14,new epithelial tissue formation was visible on the surface of the defect in all groups,with a small amount of inflammatory cell infiltration scattered in the Model group.No granulation tissue edema was seen in any of the groups,and collagen fibres were visible,with the Blank and HB groups being the most obvious.(5)Masson staining results There was no significant difference between the Blank group and DF group.(6)TUNEL staining results On the 7th day after modeling,the apoptosis of the traumatic surface of the mice in each group was detected by TUNEL method,which was 16.67±3.78 in the Blank group,43.67±4.51 in the Model group,26.33±1.53 in the HB group and 32.33±5.03 in the DF group.DF groups were statistically different(P < 0.05),and the compound cypress liquid coating could reduce epidermal cell apoptosis in mice.(7)Results of vascularization of mice trauma The number of new blood vessels in the HB group was better than that in the Blank group and the DF group(P<0.05),indicating that the compound cypress solution has a better effect on promoting wound healing.better effect on promoting wound healing.(8)Expression of proliferation of Ki67 cells in mice The expression level of Ki67 antigen was 27.67±2.52 in the Blank group,10.67±2.08 in the Model group,43.33±4.72 in the HB group and 29±3.61 in the DF group on day7.The difference was statistically significant(P<0.05).(9)Expression of IL-1 protein in mice The protein level of IL-1 in the traumatized cells of each group was measured on the7 th day of modeling: 5.67±2.08 in the Blank group,20.67±3.05 in the Model group,11.67±1.52 in the HB group and 13.67±1.53 in the DF group and not significantly different from Blank(P > 0.05).4.Conclusion(1)At sptcific drug concentrations,E.coli and its biofilm were significantly inhibited by Phellodendron cypress liquid coating,and the inhibitory effect was enhanced with increasing drug concentrations.(2)Compound Huangbai liquid coating affects biofilm formation by upregulating the transcription of mar A and omp F.(3)The compound Phellodendron Bark extract not only has good anti-infective and antibacterial effects on the infected wounds formed by E.coli and its biofilm,but also promotes the healing of the wounds and shortens the healing time. |
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