Curcumin Protects Against Liver Fibrosis In Mice By Attenuating The Infiltration Of Monocytes-derived Macrophages

BACKGROUNDLiver fibrosis is a reversible wound-healing response characterized by the accumulation of extracellular matrix (ECM). Liver fibrosis is a serious public health problem which may progress to cirrhosis, liver cancer and even death. Thus, elucidating the mechanisms of liver fibrosis and developing antifibrotics that prevent the progression toward cirrhosis are urgently needed.Previous studies suggest that hepatic stellate cells (HSC) play a key role in the pathogenesis of liver fibrosis. HSC was considered as the central regulator of liver fibrosis. Therefore, the study of the mechanisms of liver fibrosis has long focused on the HSC. However, in recent years, more and more studies have shown that monocytes/macrophages are involved in liver inflammation and fibrosis, and play a more critical role in liver fibrosis. Macrophages can participate in liver fibrosis through a variety of ways. Thus, macrophages are considered to be the center regulator of liver injury and fibrosis.During experimental tissue injury, expansion in macrophage numbers occurs via proliferation of the resident population that characterizes the later phases of inflammatory response when tissue repair and regenerative responses prevail. More importantly, circulating monocytes can be recruited to inflamed liver tissue and differentiate into macrophages. However, infiltrating monocytes in patients with chronic liver diseases (CLD) are not well characterized.Monocyte chemoattractant protein-1 (MCP-1) plays a key role in the infiltration of monocytes to the liver tissue. The chemokine (C-C motif) ligand 2 (CCR-2) is the ligand of MCP-1, MCP-2 and MCP-3, which mainly expressed on the surface of monocytes. The MCP-1/CCR-2 interaction may play an important role in recruitment of peripheral blood monocytes to the sites of inflammation liver.Animal studies have shown that the hepatic monocyte-derived macrophages play a key role in the occurrence of liver fibrosis, and MCP-1 is an important chemokine which plays a vital role in the monocyte infiltration. However, whether MCP-1 mediates the infiltration of monocytes into the liver tissue of CLD patients still needs further investigation.While preclinical development of potential anti-fibrotics is far advanced, with numerous pharmacological promising agents, few have entered clinical validation. In view of the central regulatory role of MCP-1 mediated monocytes infiltration in the pathogenisis of liver injury and fibrosis, inhibiting MCP-1 and thus reducing the infiltration of monocytes in liver tissue have become a novel target of anti-fibrotic strategy.Curcumin is a natural polyphenolic compounds extracted from plants of the genus turmeric, turmeric and Curcuma rhizome. Many animal experiments show that curcumin has good anti-fibrotic effect. But its mechanisms are not fully understood. We hypothesis that curcumin may protect the liver from liver injury and fibrosis by attenuating the recruitment of Gr1hi monocytes into liver through inhibition of MCP-1.Liver tissue samples were collected form CLD patients with different etiologies. Hepatic macrophages and monoctye-derived monocytes as well as MCP-1 expression were determined by immunohistochemistry in the paraffin-embedded liver tissues. Using carbon tetrachloride (CCl4)-induced liver damage and liver fibrosis mouse model, the role of monocyte-derived macrophages in liver injury and fibrosis were investigated. The anti-hepatitic and anti-fibrotic effects of curcumin were explored. We further investigated whether curcumin might protect the liver from CCl4-induced liver injury and fibrosis by attenuating the recruitment of Gr1hi monocyte-derived macrophages into CCl4-injured liver in mice through inhibition of MCP-1. These new findings may extend our understanding on the mechanisms of the anti-inflammatory and antifibrotic effects of curcumin and provide a novel approach for pharmacological treatment of liver fibrosis.METHODSPart I Infiltration of monocytes-derived macrophages in the liver tissue of patients with chronic liver diseasesNinety-three paraffin-embedded liver specimens from 93 CLD patients including chronic hepatitis B (CHB, n=65), autoimmune hepatitis (AIH, n=14), alcoholic liver disease (ALD, n=7) and primary biliary cirrhosis (PBC, n=7) were included in the present study. Twenty-three healthy liver tissue samples (HC) were obtained from donors whose livers were used for transplantation. Hepatic macrophages and monocytes-derived macrophages as well as the MCP-1 expression in liver tissue of CLD patients were determined by immunohistochemistry. Differences of hepatic CD68+ macrophages and MAC387+ monocyte-derived macrophages as well as MCP-1 expression in liver tissue between CLD patients and normal control were compared. Hepatic CD68+ macrophages and MAC387+ monocyte-derived macrophages as well as MCP-1 expression in liver tissue were compared in CLD patients with different inflammatory grades (G) and fibrotic stages (S).Part Ⅱ Curcumin protects against acute liver injury and liver fibrosis in mice by inhibiting monocyte-derived macrophage infiltration1. Curcumin protects against acute liver injury in mice by inhibiting monocyte-derived macrophage infiltrationC57BL/6 mice were intraperitoneally injected with CCl4 a single time to induce acute liver injury model. Curcumin was administrated orally. After 48 h of CCl4 injection, mice were sacrificed. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were dectected in each group. HE staining was performed in the liver tissue of each mouse. Percentages of immune cells including T cells, dendritic (DC) cells, natural killer (NK) cells, NKT cells and monocyte-derived macrophages subsets in the liver tissue were assessed by flow cytometry. CD45+ leukocytes, F4/80+ macrophages and CD11b+ monocytes infiltration were determined in the liver tissue of each group by immunohistochemistry. The inflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β mRNA expression in the liver tissue was dectceted by Real time PCR. The expression of MCP-1 in liver tissue was determined by Real time PCR and immunohistochemistry.2. Curcumin protects against liver fibrosis in mice by inhibiting monocyte-derived macrophage infiltrationC57BL/6 mice were injected intraperitoneally with CCl4 twice weekly for 6 weeks to establish liver fibrosis model. Curcumin was oral administrated for 6 weeks. Serum ALT and AST levels were detected. HE and Masson staining were performed in the liver tissue. Percentages of T cells, DC cells, NK cells, NKT cells and monocyte-derived macrophage subsets in the liver tissue were detected by flow cytometry. CD45+ leukocytes, F4/80+ macrophages and CD11b+ monocytes infiltration was determined in the liver tissue of each group by immunohistochemistry. TNF-a, IL-6, IL-1β and TGF-β1 mRNA expression in the liver tissue was detected by Real time PCR. The expression of MCP-1 in liver tissue was determined by Real time PCR and immunohistochemistry. a-SMA immunohistochemistry was performed in liver tissue to assess the HSC activation.RESULTSPart I Infiltration of monocytes-derived macrophages in the liver tissue of patients with chronic liver diseases1. Hepatic CD68+ macrophages were significantly increased in the liver tissue of CLD patients including CHB, PBC, AIH and ALD as compared with normal liver tissue. No difference of hepatic CD68+ macrophages was found among the liver tissue of CHB, PBC, AIH and ALD patients. Hepatic CD68+ macrophages in the liver tissue of CLD patients were significantly increased with G grades. The numbers of hepatic CD68+ macrophages was significantly increased in CLD patients with different fibrotic stages as compared with the control group. However, the numbers of hepatic CD68+ macrophages was not different in the liver tissue of CLD patients with different fibrotic stages. The numbers of hepatic CD68+ macrophages in patients with CLD were significant positively correlated with ALT, AST, gamma glutamyl transferase (GGT), total bilirubin (Tbil) and Child-Pugh score.2. The numbers of MAC387+ monocyte-derived macrophages were significantly increased in the livers of CLD patients with different etiologies as compared with the control group. However, no difference was found among CHB, PBC, AIH and ALD regarding the numbers of MAC387+ monocyte-derived macrophages. MAC387+ monocyte-derived macrophages were significantly increased in CLD patients with different G grades. CLD patients with G4 grade had significantly more MAC387+ monocyte-derived macrophages in the liver tissue as compared with Gl, G2 and G3 grades. The numbers of MAC387+ monocyte-derived macrophages were significantly increased in the liver tissue of CLD patients with different fibrotic stages. While CLD patients with S4 stage had significantly more MAC387+ monocyte-derived macrophages in the liver tissue as compared with SO-1, S2 and S3 stages. MAC387+ monocyte-derived macrophages in the liver tissue of CLD patients were not correlated with ALT, AST, GGT, Tbil and Child-Pugh. The numbers of MAC387+ monocyte-derived macrophages were negatively correlated with albumin (Alb) and positively correlated with C-reaction protein (CRP) levels.3. In the normal liver tissue, almost no expression of MCP-1 was found. However, MCP-1 expression was significantly increased in the liver tissue of CLD patients. MCP-1 staining scores in the CHB, PBC, AIH and ALD patients were significantly higher than the normal control group. Patients with CHB and ALD had relatively higher MCP-1 staining scores than PBC and AIH patients. Consistent with the MAC387+ monocyte-derived macrophages, patients with higher G scores and S scores had significantly higher MCP-1 staining scores than those patients with lower scores. Correlation analysis showed that the expression of MCP-1 was positively correlated with the numbers of hepatic CD68+ macrophages and MAC387+ monocyte-derived macrophages.Part II Curcumin protects against acute liver injury and liver fibrosis in mice by inhibiting monocyte-derived macrophage infiltration1. Curcumin protects against acute liver injury in mice by inhibiting monocyte-derived macrophage infiltrationSingle CCl4 injection could significantly cause acute liver injury. Serum ALT andAST levels in the CCl4 injected mice were significantly higher than normal controls. HE staining showed that CCl4 injection resulted in periportal necrosis and accompanied by a massive infiltration of leukocytes into the liver. Curcumin treatment did not significantly reduce serum ALT and AST levels, but can significantly reduce liver cell damage and inflammatory cell infiltration. Monocyte-derived macrophages in the liver tissue of mice were significantly increased after a single intraperitoneal injection of CCl4 as compared with the control group, while the increases of monocyte-derived macrophages were mainly Gr1hi monocyte-derived macrophages. However, other immune cells such as T cells, DC cells, NK cells and NKT cells did not change significantly. The curcumin can inhibit the infiltration of monocyte-derived macrophage, specially the Gr1hi monocyte-derived macrophages. Immunohistochemical analysis showed the numbers of CD45+ leukocytes, F4/80+ macrophages and CD11b+ monocytes increased significantly in the model group as compared with the control group. However, curcumin can significantly reduce the infiltration of these cells. The expression of inflammatory cytokines TNF-α and IL-6 in liver tissue was significantly increased in the model group after 48 h of CCl4 injection than normal control group. IL-1βhas also tended to increase. TNF-a, IL-6 and IL-1β expression in liver of curcumin treated mice was not significantly reduced as compared with the model group. The MCP-1 mRNA expression in liver tissue of model group was significantly increased as compared with normal control group. The MCP-1 mRNA level in the liver tissue of curcumin group was also significantly higher than the control group. But no significant decrease was found as compared with the model group. MCP-1 immunohistochemical analysis indicated MCP-1 staining was significantly enhanced in the liver tissue of the model group as compared with the control group, while curcumin can significantly reduced the MCP-1 expression.2. Curcumin protects against liver fibrosis in mice by inhibiting monocyte-derived macrophage infiltrationRepeated CCl4 injections could cause significant liver fibrosis in mice. Serum ALT and AST levels were significantly higher in the model group than normal control group. Massive necrosis of liver cells, lobular structural disorder and massive inflammatory cell infiltration were found in the liver tissue of CCl4 treated mice by HE staining. Masson staining showed a high level of collagen deposition. Curcumin treatment could significantly reduce serum ALT and AST levels in mice and reduced liver cell damage, inflammatory cell infiltration and liver fibrosis. Monocyte-derived macrophages eapecially the Gr1hi monocyte-derived macrophages were significantly increased in the liver tissue of CCl4 treated mice as compared with the control group. The proportions of CD3+T cells and DC cells were significantly higher in the model group than the control group, while NK and NKT cells were significantly lower than the control group. The curcumin had no significant effect on T cells, DC cells, NK cells and NKT cells. However, the curcumin could inhibit the infiltration of monocyte-derived macrophage, specially the Gr1hi monocyte-derived macrophages. Immunohistochemical analysis showed the CD45+ leukocytes, F4/80+ macrophages and CD11b+ monocytes were increased significantly in the model group as compared with the control group, while the curcumin could significantly reduce the infiltration of these cells. After six weeks of CCl4 injection, the TNF-a expression was significantly increased in the liver tissue of model group than normal control group, while the IL-1β and IL-6 mRNA expression was also found an increasing trend. Curcumin significantly reduced the proinflammatory cytokine TNF-α in mRNA levels in liver tissue. Within the liver tissue of model group, the TGF-β1 mRNA levels were significantly higher than normal control group. Curcumin could significantly reduce the TGF-β1 mRNA in the liver tissue as compared with the model group. Real time PCR and immunohistochemistry indicated that the MCP-1 expression in the liver tissue of model group was significantly higher than that of control mice. The curcumin significantly reduced MCP-1 expression in mouse liver tissue.α-SMA immunohistochemistry suggested curcumin could significantly inhibit the HSC activation.CONCLUSIONS1. The hepatic macrophages and monocyte-derived macrophages were significantly increased in the liver tissue of CLD patients with different etiologies as compared with normal control and were correlated with the degrees of inflammation and fibrosis. MCP-1 may be involved in monocyte-derived macrophages infiltration in CLD patients.2. Curcumin has anti-inflammatory and antifibrotic effects. The anti-inflammatory and antifibrotic effects of curcumin could be contributed to its prevention of Gr1hi monocyte infiltration into the injured livers through inhibition of MCP-1.