Ghrelin's second life - World Journal of Gastroenterology

A
MCP-1 expression (fold change)
8
7
6
5
4
3
2
1
0
Control
Protein
Gene
CCl4-7 wk
CCl4-13 wk
marrow to differentiate into hepatocytes. To assess the
possibility of YGJ stimulating bone marrow cells to
differentiate into hepatocytes, we investigated the presence
of EGFP + /Alb + cells by double staining. In the
CCl4­injected control mice, the number of EGFP + cells
increased steadily over time and the cells were distributed
mainly in mesenchymal areas, but Alb + cells were
mainly distributed in parenchymal areas, and there was no
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CCl4-YGJ
B 1 2 3 4
C
Wang XL et al . YGJ decoction protects against hepatic injury
MCP-1
GAPDH
CCR2 expression (fold change)
CCR2
6
5
4
3
2
1
0
Control
Protein
Gene
b
a
CCl4-7 wk
a
b
a
c
c
26 KD
42 KD
36 KD
Figure 7 Gene expression analyzed by real-time polymerase chain reaction
and protein expression analyzed by Western blotting of monocyte
chemotaxis protein-1 and CC chemokine receptor 2 in CCl4-induced
chronic liver injured bone marrow chimera mice. A: MCP-1 gene and protein
expression. For both genes the relative expression is quantified using the control
group as the basal level; B: MCP-1 and CCR2 bands by Western blotting. Band
1, control group; band 2, CCl4 7 wk group; band 3, CCl4 13 wk group; and band 4,
CCl4 + Yiguanjian (YGJ) group; C: CCR2 gene and protein expression. For both
genes the relative expression is quantified using the control group as the basal
level. MCP-1: Monocyte chemotaxis protein-1; CCR2: CC chemokine receptor 2;
GAPDH: Glyceraldehydes-3- phosphate dehydrogenase. a P < 0.05, b P < 0.01 vs
week 0 control group; c P < 0.05 vs the same time-point CCl4 group.
b
a
CCl4-13 wk
c
CCl4-YGJ
overlap between EGFP + cells and Alb + cells, indicating
that no EGFP + /Alb + cells were present in chronically
injured liver. No EGFP + /Alb + cells were present in the
vehicle control mice or in the YGJ­treated mice (Figure
5). These results seemingly suggest that no hepatocytes
were derived from bone marrow in our study.
To further investigate the mechanisms of YGJ mediation
of bone marrow cell differentiation in chronic liver
injury, we explored the possibility that BM cells differentiated
into other main types of cells in the liver, such
as Kupffer cells. The EGFP + /F4/80 + cells represented
BM­derived Kupffer cells in our experiments. In the vehicle
control mice, despite of a little F4/80 expression,
virtually no EGFP + /F4/80 + cells were present in the liver.
However, after 7 wk of CCl4 injection, coexpression
of EGFP and F4/80 increased significantly (P < 0.01)
and remained at a high level up to 13 wk. These results
indicated that some Kupffer cells differentiated from
BM cells as the hepatic injury progressed. With YGJ
treatment, both EGFP and F4/80 were expressed in parenchymal
areas, and some of the areas were overlapped
(Figure 6). However, there were fewer EGFP + /F4/80 +
cells in the YGJ-treated mice than in the CCl4-injected
mice (P < 0.01).
YGJ decoction suppresses monocyte chemotaxis
protein-1 and CCR2 expression in fibrotic liver
MCP­1 was identified as a factor likely to be responsible
for stem/progenitor cell recruitment. Therefore, we
analyzed the expression of MCP­1 during CCl4-induced
fibrogenesis. For the gene and protein expression, results
were normalized to expression in the liver at the time
of CCl4 injection (0 wk), for example the control group
mRNA level of MCP­1 reached 2.8­fold after 7 wk (P
< 0.05) and 6.7­fold after 13 wk of CCl4 injection (P <
0.05) (Figure 7A). Western blotting showed that MCP­1
protein was increased markedly (approximately 4 fold)
after 13 wk of CCl4 injection (Figure 7A and B). YGJ
treatment significantly reduced the expression of both
MCP-1 gene and protein (Figure 7A and B). CCR2 is the
specific receptor of MCP­1, and both Kupffer cells and
HSCs, but not hepatocytes, have been shown to express
CCR2 in liver [16] . In our study, the gene expression of
CCR2 continuously increased over the course of the
experiment (P < 0.05), which was in accordance with its
protein expression (P < 0.01), and treatment with YGJ
only suppressed expression at the protein level (P < 0.05)
but not at the gene level (Figure 7B and C). These data
demonstrate that more exogenous stem/progenitor cells
were recruited into chronically injured livers compared
with the controls, but that YGJ treatment decreased the
number of exogenous stem/progenitor cells in the liver.
YGJ decoction decreases proliferation of liver epithelial
progenitor cells and mature hepatocytes
It is important to determine whether the proliferative
activity of hepatocytes increased during hepatic injury
since the hepatocytes are the most numerous cells in
3244 July 7, 2012|Volume 18|Issue 25|