Effect Of Mitochondrial Dynamics On Macrophage Polarization In Hepatic Alveolar Echinococcosis

Objective: We analyzed the changes of the expression levels of mitochondrial dynamics and macrophage polarization markers after echinococcus multilocularis infection at the cell and human to explore the effect of mitochondrial dynamics on the macrophage polarization in hepatic alveolar echinococcosis.Methods: At the tissue level,RNA-seq was used to detect the expression levels of mitochondrial dynamics and macrophage polarization related markers in marginal zones,lesions and normal liver tissues of 4 patients with advanced hepatic alveolar echinococcosis.Immunohistochemistry and immunofluorescence labeling related proteins.At the cell level,the echinococcus multilocularum was co-cultured with RAW264.7 at four time points of 1,3,5 and 7 days.Cell morphology was observed by inverted microscope.Gene expression levels of mitochondrial dynamics and macrophage polarization markers were detected by quantitative real-time PCR,and Western blot.The graph was made by Graph Pad Prism 8.0 software,and statistical analysis was performed by Student’s t test.P < 0.05 showed a significant difference,while ns showed no significant difference.Results: At the tissue level: RNA-seq results showed the expression of MFN2 in the marginal zones was higher than normal(P=0.0230).FIS1 expression was significantly increased in the marginal zones(P=0.0154),and the expression in the lesions was not significant compared with that in the normal control group.The expression of TGF-β and M-CSF was up-regulated in the lesions(P=0.0350,P=0.0384).ARGINASE was down-regulated in the lesions(P=0.0093).Immunohistochemical results: The expression of OPA1 in the lesions was down-regulated(P=0.0124).The expression of FIS1 was up-regulated in the marginal zones(P=0.0368)and downregulated in the lesions(P=0.0011).Immunofluorescence results showed that the expressions of MFN1/2,OPA1 and FIS1 were not significant compared with that in normal liver tissues.The expression of DNM1 in the lesion tissue was up-regulated compared with that in the normal liver tissues(P=0.0157).At the cell level,microscopic observation of RAW264.7 in normal culture showed spherical or droplet shape,with a few pseudopods protruding and short length.When RAW264.7 co-cultured with the echinococcus multilocularis was stimulated,the number of pseudopods in most cells increased and the length of the pseudopods was significantly longer than that of normal cultured cells,and it even connected with the extended pseudopods of neighboring cells to establish intercellular connections.Quantitative real-time PCR results showed that the expression level of mitochondrial fusion gene OPA1 in the experimental group was significantly higher than that in the normal cells at the first and third day(P < 0.0001,P=0.0095).On the first day,the expression level of MFN1 was higher than that of the normal group(P < 0.0001).However,the expression level of MFN2 decreased significantly on the third day(P=0.0040)and increased on the seventh day(P < 0.0001).The expression of FIS1 showed a trend of increasing on the first day(P=0.0338).The expression of DNM1 in the control group was higher than that in the normal control group during the whole co-culture cycle,with statistical significance at the 1st,3rd and7th(P=0.0247,P=0.0151,P=0.0017).The results of the detection of polarization related genes in macrophages showed that the M1-like polarization gene IFNg-R1 showed an upward trend on the third day(P=0.0153),and decreased on the first and seventh days(P=0.0470,P=0.0025).The expression of IFNg-R2 decreased significantly on the 5th and 7th days(P=0.0232,P=0.0257).TNF-α increased at 1,3 and 7 days(P < 0.0001,P=0.0002,P=0.0006);IL12P40 increased on day 1,3 and 5(P=0.0163,P=0.0053,P=0.0105).TGF-β showed an increasing trend on day 1(P=0.0015),and decreased on day 5 and 7(P=0.0471,P=0092).The expression of Arginase increased throughout the whole cycle(P=0.0007,P=0.0245,P < 0.0001,P=0.0332).The expression of M-CSF increased on day 3 and 5(P=0.0236,P=0.0006).The expression level of GM-CSF in the experimental group was higher than that in the control group throughout the whole cycle(P=0.0064,P=0.0288,P=0.0004,P=0.0006).Western blot results showed that the expression of mitochondrial fusion protein MFN1/2 was up-regulated on the first day,and decreased on the third,fifth and seventh days.The expression of OPA1 was upregulated on the first and third days,and decreased on the fifth and seventh days.The expression of mitochondrial fission protein FIS1 expression was up-regulated on day 1and 7,and decreased on day 3 and 5.Conclusions: Infection with echinococcus multilocularis causes mitochondrial dynamic changes in vivo and in vitro,with mitochondrial fission as the dominant role.At the same time,the dynamic balance of polarization of M1 and M2 macrophages was broken,and their combined effect promoted the development of hepatic alveolar echinococcosis.