| Pseudomonas aeruginosa is widely distributed in the natural environment and is one of the common pathogens in the soil.It is also one of the three most easily infected pathogens in the hospital.Pseudomonas aeruginosa can secrete a variety of secondary metabolites and is considered to be an important biological control bacteria.Its secondary metabolites are not only found to have broad-spectrum antibacterial activities(such as phenazines),but also considered to inhibit inflammation and potential for cell proliferation(such as cyclic dipeptides).The secretion of secondary metabolites of Pseudomonas aeruginosa is closely related to the bacterial biofilm(BF),and is also related to the quorum sensing system(QSS)and type VI secretion system(T6SS).Therefore,genes that regulate the synthesis and secretion of secondary metabolites may affect the BF,QSS and T6 SS related gene expression.In this experiment,a lipopolysaccharide-induced RAW264.7 mouse macrophage inflammation model was established,and explored the chloroform extract of Pseudomonas aeruginosa(CEPA)and the purified product(1-hydroxyphenazine)in vitro antiinflammatory activity.At the same time,the experiment explored the effect of phz R gene on the expression of genes related to BF,QSS and T6 SS,using the wild strain of Pseudomonas aeruginosa 2016NX1 and the constructed high-yielding phenazine strain 2019NES(highly expressing the phz R gene).The methods and results are as follows:1.The MTT method was used to detect the cell survival rate,and determine the safe concentration of LPS,CEPA and 1-hydroxyphenazine on the survival rate of RAW264.7cells for subsequent experiments.The results showed that LPS at 0～10 μg/m L,CEPA at0～20 μg/m L,and 1-hydroxyphenazine at 0～5 μg/m L have no significant effect on cell viability and are safe concentrations for cells.2.The effects of CEPA and 1-hydroxyphenazine on the level of TNF-α secreted by cells were measured by ELISA.The results showed that both can inhibit the secretion of TNF-α in a concentration-dependent manner.The higher the drug concentration,the more obvious the inhibitory effect.3.Morphological observation method was used to determine the changes in cell morphology after CEPA and 1-hydroxyphenazine treatment.It was found that when only LPS stimulated,the cell volume increased and the pseudopodia protruded.And CEPA or 1-hydroxyphenazine was added to the treatment together with the drug,as the concentration increases,the cell volume decreases,the pseudopodia become shorter,there is a tendency to return to normal cell morphology.4.RT-q PCR method detected the changes in the expression levels of inflammatory factors TNF-α,IL-1β and IL-6 m RNA.The results showed that CEPA and 1-hydroxyphenazine can inhibit TNF-α,IL-1β and IL-6 m RNA.The relative expression of TNF-α,IL-1β and IL-6 m RNA decreased gradually with the increase of the concentration of the two drugs,indicating that CEPA and 1-hydroxyphenazine have an inhibitory effect on the expression of m RNA.5.The RT-q PCR method compared the gene expression differences of bacterial biofilm(fil C,psl A,pel A,pil A),quorum sensing system(las R)and type VI secretion system(hcp1,hcp2,hcp3)between the wild strain of Pseudomonas aeruginosa 2016NX1 and the engineered strain 2019 NES.The results showed that only las R and pil A were significantly different in the planktonic state.The expression level of 2019 NES is lower than 2016NX1.There are significant differences between las R,psl A and pel A in the envelope state.The expression level of 2019 NES is also lower.6.The crystal violet staining method was used to determine the expression of the biofilm amount of 2016NX1 and 2019 NES in the 12 h,1 d,3 d and 6 d time periods.The results showed that the amount of biofilm of the two strains increased as the culture time increased.But the amount of 2016NX1 biofilm continues to be higher than 2019 NES.7.The flat panel analysis method is used to compare the twitching,swimming and swarming abilities of 2016NX1 and 2019 NES.The results show that the twitching ability of the two is the same.The swimming ability of 2019 NES is stronger than 2016NX1,and the swarming ability of 2016NX1 is stronger than 2019 NES.In summary,CEPA and 1-hydroxyphenazine can inhibit the secretion of inflammatory factors,reduce the expression of inflammatory genes,help cells restore their normal form,and have a certain anti-inflammatory effect.At the same time,the phz R gene can affect the expression of genes related to bacterial biofilm,quorum sensing system and type VI secretion system,reduce the formation of bacterial biofilm,and affect the motility of bacteria. |
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