Study Of Effects Of Salmonella Enteritidis In Chicken Cell Autophagy And Its Fe₃O₄-MNP Regulation

This study was aimed to investigate the effects of Fe₃O₄ Magnetic Nanoparticles（Fe₃O₄-MNP）on Avian cell when infected with Salmonella enteritidis（SE）from the perspective of autophagy.1.Study of whether SE infection can induce autophagy in LMH cells.LMH cell was selected as our research object,and a model of SE infection has constructed.The occurrence of autophagy was detected by Transmission electron microscope（TEM）and Western-blot（WB）.The results showed as follows:1)When the concentration of SE was 5×10⁷ cfu/mL,and co-culture time was 1 hour,most of LMH cells were invaded by SE,but cell state not affected,no cytoplasmic loosening or cell disintegration.2)TEM results showed that compared with mock-infected cells,both double-or single-membrane autophagic vesicles were significantly increased post infection.3)WB results showed that compared with mock-infected cells,LC3-II/β-actin was significant increased in LMH cells infected by SC070（P<0.05）,while p62/β-actin significant decreased（P<0.01）.2.Study of the regulation of Fe₃O₄-MNP in SE infection.Different dose of Fe₃O₄-MNP（50,100,200,400μg/mL）were added to treat the LMH cells infected by SE,and set a control group without adding Fe₃O₄-MNP at the same time.LMH cells were collected at different hours post infection（hpi）,determining their cell viabilities by CCK-8,and counting the intracellular SE loads by plate counting method.The results showed as follows:1)At 6 hpi,compared with the control group,all doses of Fe₃O₄-MNP had no significant influence to intracellular SE loads（P>0.05）;200 and 400μg/mL Fe₃O₄-MNP reduced cell viabilities significantly（P<0.05）.2)At 12 hpi,compared with the control group,all doses of Fe₃O₄-MNP reduced intracellular SE loads very significantly（P<0.01）;100μg/mL Fe₃O₄-MNP had a trend of increasing cell viability（P>0.05）,50and 200μg/mL Fe₃O₄-MNP had no significant influence to cell viabilities（P>0.05）,400μg/mL Fe₃O₄-MNP reduced cell viability significantly（P<0.05）.3)At 18 hpi,compared with the control group,all doses of Fe₃O₄-MNP reduced intracellular SE loads very significantly（P<0.01）,50μg/mL Fe₃O₄-MNP had no significant influence to cell viabilities（P>0.05）,100,200 and 400μg/mL reduced cell viabilities very significantly（P<0.01）.4)At 24 hpi,compared with the control group,all doses of Fe₃O₄-MNP reduced intracellular SE loads very significantly（P<0.01）.5)At 30 hpi,compared with the control group,all doses of Fe₃O₄-MNP reduced intracellular SE loads and cell viabilities very significantly（P<0.01）,and with the increase of dose,the intracellular SE loads and cell viabilities decreased.3.Study of the effects of autophagy in regulation of Fe₃O₄-MNP in SE infection.100μg/mL Fe₃O₄-MNP was added to treat the LMH cells infected by SE（group SN）,and set a control group without adding Fe₃O₄-MNP（group SE）at the same time.LMH cells were collected at 12hpi,determined autophagy of the two groups by TEM and WB.The results showed that compared with group SE,autophagic vesicles in LMH cells of group SN were increased,and LC3-II/β-actin significant increased（P<0.05）,p62/β-actin significant decreased（P<0.05）.In conclusion,in our experiments,the suitable dosage for constructing SE infection model was5×10⁷ cfu/mL,and suitable co-culture time was 1 hour;SE infection could induce autophagy in LMH cells;Fe₃O₄-MNP could alleviate SE infection of LMH,and effects of concentration at 100μg/mL for12 hours was the best than the others;Fe₃O₄-MNP alleviate SE infection of LMH by promoting autophagy.