Effect Of Autophagy On Macrophage Polarization In Hepatic Alveolar Echinococcosis

Objectives: To explore whether there is autophagy and macrophage polarization in hepatic vesicular hydatid disease,and the possible role of autophagy and macrophage polarization in the process of echinococcus multilocularis infection,and then to study whether autophagy has an effect on macrophage polarization.Methods: The mouse bone marrow-derived macrophages RAW264.7 were cocultured with the protocrops of echinococcus multilocularis,and four time nodes were selected for the experiment,the first day,the third day,the fifth day,and the seventh day.Observe the changes of cell morphology by microscope,observe autophagosomes by transmission electron microscope,and detect the expression of autophagy and macrophage polarization markers from two levels of gene and protein by real-time quantitative PCR detecting system and western blotting.Select the focal tissue,marginal zone tissue and normal liver tissue of patients with hepatic alveolar echinococcosis,and detect the expression of autophagy and macrophage polarization markers at the two levels of genes and proteins by NGS,immunohistochemistry,and immunofluorescence.Graphpad Prism 8.0 was used for graphing,and statistical analysis was performed by two-tailed t test(Student’s t test);P<0.05 has a significant difference.Results: Human tissue level: The expression of mitochondrial autophagy-related genes PINK1 and PARK2 in the lesion and marginal zone was lower than that of the control group.The expression of autophagy-related gene LC3 B in the lesion(P=0.0401)and marginal zone were higher than those in the control group.The expressions of M1 macrophage related genes IFNGR1 and IFNGR2 in the lesion and marginal zone were higher than those in the control group.The expression of M2 type macrophage-related genes TGFβ and MCSF in the lesion and marginal zone was higher than that in the control group,while the expression of ARGINASE in the lesion and marginal zone was decreased.The results of immunohistochemistry showed that the expression of PINK1 and PARKIN in the lesion was lower than that in the control group;LC3B showed that the expression in the lesion group was lower than that in the control group,but the expression in the marginal zone was higher than that in the control group(P=0.0264).Immunofluorescence showed that the expression of PINK1 in the marginal zone was higher than that in the control group,and the expression in the lesions did not change significantly compared with the control group,and both were not statistically significant;The expression of PARKIN in the marginal zone and the lesion group was lower than that of the control group,and the lesion group was statistically significant(P=0.0104);The expression of LC3 B increased in the marginal zone and decreased in the lesion group,but there was no statistical significance.Cell level: After RAW264.7is co-cultured with echinococcus multilocularis protocrops,the morphology of the cells under the microscope changes.With the extension of the co-cultivation time,the morphology gradually changes from round and quasi-circular to irregular.Extend the pseudopod and establish contact with surrounding cells.Under the transmission electron microscope,autophagosomes coated with a double-layer membrane can be observed in a round or round-like shape.The q PCR results suggested that the mitochondrial autophagy-related genes Pink1 and Park2 showed an overall upward trend,but there was no significant statistical significance.M1 type macrophage-related genes showed an overall increasing trend on the first and third days,and partly increased and partly decreased on the fifth and seventh days.The overall expression of M2 macrophage-related genes during the entire co-culture process was higher than that of the control group.The ratio of the gray value of the autophagy protein LC3 B band to the internal control band was higher than that of the control group on the 3rd,5th,and 7th day.The ratio of the gray value of the mitochondrial autophagy protein PINK1 and PARKIN band to the internal reference protein band was lower than that of the control group at the four time points.Conclusions: 1.In hepatic alveolar hydatid autophagy and macrophage polarization are simultaneously present in the lesion and the marginal zone of the patient;2.Under the stimulation of the protocranial segment of echinococcus multilocularis,autophagy and polarization occurred in RAW264.7,and the macrophages of the two subtypes were in a state of dynamic transformation.