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.