The Effects Of Peroxisome Proliferator-activated Receptors On Macrophage Polarization In Alveolar Echinococcosis

Objective 1.To explore the effects of PPARs expression in macrophage polarization in alveolar echinococcosis by co-culture of RAW264.7 with multilocular echinococcus protoscolex(PSC)in vitro.2.To verify the effect of PPARs expression on macrophage polarization with alveolar echinococcosis patients’ liver tissue.Methods 1.The morphological changes of macrophages were observed on day 1,3,5 and 7 after co-culture with protoscolex and the cell length and area were measured.2.The mRNA and protein expression of PPARs(α,β/δ,γ)in macrophages were determined by qRT-PCR and Western Bolt(WB)on day 1,3,5 and 7 after co-culture with protoscolex.3.RAW264.7 macrophages were co-cultured with protoscolex and macrophages were collected on day 1,3,5,7.The mRNA expression of M1 macrophages-associated markers including TNF-α、IFN-γ R1、GM-CSF,and M2 macrophages-associated markers including Arg-1、TGF-β、M-CSF was detected by qRT-PCR.4.The effects of drug intervention(PPARα,β/δ,γ agonist or antagonist)on macrophage polarization were observed after co-culture of RAW264.7 and protoscolex.5.The areal densities of M1(CD86)and M2(CD206)macrophages in normal liver tissues and liver lesion ranging were measured by immunofluorescence in 8 patients with alveolar echinococcosis.6.Immunofluorescence was used to detect the average intensity of PPARs in CD86 and CD206 macrophages with alveolar echinococcosis patients’ liver tissue.Results 1.Microscope observation showed that macrophage morphology was diverse and irregular with pseudopodia formation during co-culture on day 1,3,5 and 7.And it was most obvious on day 7.After measuring the length and area of macrophages,it was found that with the increase of co-culture time,the length of macrophages became significantly longer,and the area showed little difference between day 1 and day 3,and increased significantly on day 5 and day 7.2.The expression of PPARβ in the macrophages co-cultured with protoscolex was also increased first and then decreased by qRT-PCR and WB,and the expression of PPARβ mRNA was the highest on day 3.The mRNA expression of PPARγ showed an increasing trend,and the expression of PPARγ mRNA was highest on day 7 after RAW264.7 co-cultured with PSC.The expression of PPARβ and PPARγ protein was basically consistent with the mRNA transcription level,which was detected by qRT-PCR and WB.PPARα expression was not observed by qRT-PCR and WB.3.After qRT-PCR detection of M1/M2 macrophage-associated markers,the M1macrophage-associated markers showed first increasing and then decreasing,but the M2 macrophage-associated marker were generally increased.The results indicated that the polarization of macrophages co-cultured with protoscolex was mainly M1 in the early stage and M2 in the late stage.4.The expression of PPARβ in RAW264.7 macrophages and M1macrophage-associated markers showed an upward and then downward trend,and their expression trend was consistent.The expression of PPARγ and M1macrophage-associated markers showed an increaing trend,and their expression trend was consistent.It was considered that PPARβ may mainly regulate the M1 macrophages polarization,PPARγ mainly regulate the M2 macrophages polarization.5.After intervention with PPARβ antagonist,the expression of M1 marker TNF-αwas significantly inhibited.The expression of M2 marker(Arg-1,M-CSF)was significantly inhibited after PPARγ antagonist intervention.These results furtherly confirmed that PPARβ may mainly regulate the M1 macrophage polarization process,and PPARγ may mainly regulate the M2 macrophage polarization process.6.It has been showed that the positive distribution of CD86 in the liver lesion ranging was smaller than that in the normal liver tissue,and the positive distribution of CD206 in the liver lesion ranging was larger than that in the normal liver tissue by immunofluorescence staining.The results suggested that macrophages in the liver lesion ranging were main polarization to M2 in the late stage of alveolar echinococcosis.7.The M1,M2 macrophages and PPARβ in the normal liver and liver lesions ranging were simultaneously stained by immunofluorescence.By analyzing the average intensity of PPARβ expression in M1 and M2 macrophages,it was found that the PPARβ expression in M1 macrophages was significantly decreased compared with that in normal liver,and the expression of PPARβ in M2 macrophages was not significantly different from that in normal liver.PPARγ expression in M2(CD206)illustrated higher PPARγ expression in the liver lesion ranging than the normal liver.The results also suggest that PPARβ may mainly regulate the M1 macrophage polarization,and PPARγ may mainly regulate the M2 macrophage polarization in alveolar echinococcosis.Conclusions In alveolar echinococcosis,both M1 and M2 macrophages are involved in the process of multilocular echinococcus infection.After the infection of multilocular echinococcus,macrophages were mainly polarized to M1,which cleared the pathogen by producing pro-inflammatory cytokines.With the extension of infection time,macrophages were polarized to M2,which maintained the continuous infection by producing anti-inflammatory cytokines.PPARβ,γ may co-regulate the polarization of macrophages in AE,and regulate M1 and M2 polarization respectively.