Microplastics Effect On Planarian Infection Pathogenic Bacteria And Innate Immune Mechanism

Background: Microplastics(MPs,Microplastics)refer to the degradation of plastics in the environment by factors such as light and heat,weakening the completeness of the plastics and causing them to split into small fragments.MPs are widespread in the environment,including water,air,and soil.And high levels of MPs have been detected in people’s diet and bottled drinks.The damage of MPs to human body includes immunotoxicity,genotoxicity,oxidative stress,inflammation,intestinal microflora disorder,etc.MPs can also be used as a carrier for microorganisms to enter the body and cause damage.Planarians,as a new animal model,have the advantages of fast reproduction and low maintenance cost,and are becoming more and more popular in the field of immune mechanism research.Many genes and proteins associated with immunity have been found in planarians,such as MORN2.However,whether the co-exposure of MPs and bacteria can lead to changes in the immune system of planarians and the mechanism is still unknown.Therefore,in this study,the feeding method was used to expose planarians to MPs,to explore the possible immune mechanism response,and to provide new clues and ideas for understanding the relationship between planarians and natural immunity.Methods: 1.Feeding MPs with different concentrations and particle sizes to observe whether there is any influence on the feeding of planaria.2.Feed Staphylococcus aureus(S.aureus)and Salmonella typhimurium(S.typhi),and observe the bacterial content in different time periods(0 d,3 d,6 d and 9 d)of planarians after MPs exposure(3 d)by plate counting and q PCR method.3.q PCR was used to detect the expression of PAQR3 in planarians exposed to MPs for3 days at different time periods after feeding the bacteria.4.q PCR was used to detect the expression of autophagy related genes(Beclin1 and ATG5)in planarians after the co-exposure of MPs and bacteria,and the autophagy in planarians was inhibited by soaking 3-MA to observe whether autophagy was involved in the immune reaction caused by MPs.5.PAQR3 expression was silenced by RNAi method to observe the changes in the content of pathogenic bacteria;The regulatory relationship between PAQR3 and autophagy was also discussed.6.Transcriptome sequencing was performed on planarians of different treatment groups using the next-generation BGISEQ sequencing platform to explore the pathway through which PAQR3 regulates autophagy.Results: 1.Under particle sizes of 10um(MPs10),1um(MPs1)and 100nm(MPs0.1),MPs with different concentrations(1%,5% and 10%)were mixed into bovine liver containing edible red pigment,and no statistical difference was found in the number of different groups of red planarians.These results indicated that MPs with different particle size and concentration did not affect the feeding of planarians.2.Exposure to MPs10(10%)significantly reduced the number of prefed bacteria(S.aureus and S.typhi)in planarians on days 3 and 6 compared to the control group(P<0.001).Exposure to other particle sizes and concentrations had no effect on the bacterial content(P>0.05),and the results were verified by q PCR.3.MPs10(10%)could increase the expression of PAQR3 on 3rd day(P<0.001),and the difference decreased on 6th day but was still higher than that of the bacteria group(P<0.01).There was no significant difference in the expression of PAQR3 on 9th day(P>0.05).For S.typhimurium,the effect trend at each time point was roughly the same as that of S.aureus,with the largest difference at 3rd day and gradually decreasing until 9th day.There was no significant difference in the expression of PAQR3 in MPs10 group(P>0.05).The m RNA expression levels of Beclin1 and ATG5 in both S.aureus and S.typhi groups were significantly higher in the bacteria+MPs10 group than in the corresponding bacteria alone group,with statistical significance(P<0.05).The bacteria content in S.aureus+3-MA group was much higher than that in S.aureus+ MPs10 +3-MA group(P<0.05).The bacteria content in S.aureus+ MPs10 +3-MA group was also much higher than that in S.aureus+MPs10 group(P<0.05).The results were also consistent for S.typhi,suggesting that autophagy is involved in the immune response of planarians.The expression level of PAQR3 in each group was observed,and it was found that there was no significant difference in PAQR3 m RNA expression level of planarians when autophagy was inhibited(P>0.05).5.When PAQR3 was inhibited,the bacterial content of S.aureus+RNAi group was significantly higher than that of S.aureus group(P<0.05),and the bacterial content of S.aureus+ MPs10 +RNAi group was also significantly higher than that of S.aureus+ MPs10 group(P<0.05).The results for S.typhi were also consistent.These results suggested that PAQR3 was involved in the immune response of planarians stimulated by MPs10.The m RNA expression of Beclin1 and ATG5 in the S.aureus+ MPs10+RNAi group was significantly inhibited compared with that in the S.aureus+MPs10 group(P<0.05),and the results were also consistent for S.typhi,suggesting that PAQR3 is upstream of autophagy regulation.6.Transcriptome sequencing analysis showed that 24962 genes were expressed in S.aureus group,S.aureus+ MPs10 group and control group;A total of25011 genes were expressed in S.typhi group,S.typhi + MPs10 group and control group.Through the enrichment analysis of GO and KEGG genes,it was found that lysosome was the first enrichment in both S.aureus and S.typhi groups,which suggested that PAQR3 regulates autophagy in planarians by affecting the biological process of lysosome.Conclusion: 1.MPs did not affect the feeding of planarians.2.Co-exposure of microplastics with particle size of 10um(MPs10)and concentration of 10% with bacteria can induce the expression of PAQR3 in planarian,and affecting the infection of pathogenic bacteria in planarian..3.Co-exposure of MPs10 and bacteria can cause autophagy in planarian.4.PAQR3 gene may regulate autophagy by affecting lysosome biological processes and affect the natural anti-bacterial immunity in planarians.