3.8 The depletion of macrophage partially neutralizes the anti-fibrotic effect of PIC Ⅱ
Based on the vital role of macrophages played in the fibrotic liver of Mdr2-/- mice, we then verified that whether the depletion of macrophages will obstruct PIC Ⅱ-induced hepatoprotective effects in a negative manner. The clodronate liposome (CL), a classical scavenger agent targeting on macrophages, was used for macrophage depletion in Mdr2-/- mice treated with or without PIC Ⅱ (Fig. 8A ). Compared with the Mdr2-/- group, CL markedly increased the levels of TBA, TBIL, AST, ALT, and γ-GGT in serum, which was almost unchanged or only slightly decreased in Mdr2-/- + CL + PIC Ⅱ group in Fig. 8B andFig. S8A . We further evaluated the pathological change after macrophage clearance and observed similar changes of inflammatory infiltration, ECM deposition, and fibrous scar in the Mdr2-/- + macrophage depletion group and Mdr2-/- + CL + PIC Ⅱ group (Fig. 8C ). Meanwhile, CXCL16 was proved to decreased and not to co-located with F4/80 both in the Mdr2-/- + CL group and Mdr2-/- + CL + PIC Ⅱ group (Fig. 8D ). To better evaluate the protective effects and underlying mechanisms of PICⅡ after macrophage clearance, we also performed qPCR to detect the gene expression of interest. Although the mRNA levels of Cxcl16 was similar before or after CL treatment, the typical marker of M1 polarized macrophages, Inos , was down-regulated both in the Mdr2-/- + CL group and Mdr2-/- + CL + PIC Ⅱ group. Besides, the meaningful changes of other immune cells were also checked as following: the phagosome marker Rab7a has not been changed evidently, the cytotoxic maker of NK cells Il4 was found to slightly decreased, and the neutrophils marker Ly6g was noticed to up-regulated in Mdr2-/- + CL group while being down-regulated in Mdr2-/- + CL + PIC Ⅱ group, which might evidence that PIC Ⅱ showed an inhibitory effect on neutrophils in liver fibrotic process. Interestingly, along with the changes of immune cells makers, the fibrotic gene expression like Fn1 and Col1a1 andActa2 remains high and PIC Ⅱ could not decrease their expression once macrophage depletion in Mdr2-/- mice (Fig. 8E and Fig. S8B ). Meanwhile, the protein levels of FIBRONECTIN and COLLAGEN in Fig. 8F also showed the similar trend in above relative groups. These results suggest that depletion of macrophages at least partially neutralized PIC Ⅱ-induced hepatoprotective effects in Mdr2-/- mice.