Study On Anti-hepatitic And Anti-ifbrotic Effects Of Matrine And Its Derivative M19and Their Inhibition On Hepatic Recruitment Of Inlfammatory Monocytes

[Background]Hepatic fibrosis, a wound-healing response to chronic liver injury, is characterized by excess production and deposition of extracellular matrix (ECM), which leads to loss of liver function and disruption of liver structure. The initiation of fibrosis crucially depends on an inflammatory phase in which liver resident macrophages, Kupffer cells, are activated and release transforming growth factor-β (TGF-β) as well as other proinflammatory cytokines that activate hepatic satellite cells (HSCs). The activation and proliferation of HSCs has been well established as the central event in the development of hepatic fibrosis. Therefore activated Kupffer cells and HSCs are attractive targets for anti-fibrotic therapy based on their key role in development of hepatic fibrosis in the past decades. Recently the infiltration of blood-derived macrophages in addition to activation of classical Kupffer cells has been suggested as being essential for liver fibrogenesis, the exact phenotype of infiltrating monocyte populations and the molecular mechanisms are also elucidated. Blood monocytes are circulating precursors of tissue macrophages and can be divided into two functionally distinct subpopulations in mice:Gr1high and Grllow monocytes. Gr1high monocytes are considered precursors for macrophages and dendritic cells in inflammatory conditions, whereas Gr1low monocytes may represent steady state precursor cells for tissue macrophages. The differential recruitment of these monocyte subsets appears to be crucially controlled by chemokines released from injured tissue. Only Gr1high monocytes express C-C chemokine receptor (CCR2). Upon toxic injury, liver tissue up-regulate hepatic expression of the ligands for CCR2, monocyte chemoattractant protein1(MCP-1)/CCL2. Therefore, only inflammatory Gr1high but not Grllow monocytes are massively recruited into the liver upon toxic injury. During chronic liver damage, intrahepatic Gr1high monocyte-derived cells differentiate preferentially into inducible nitric oxide synthase-producing macrophages exerting proinflammatory and profibrogenic actions, such as promoting HSC activation, T helper1-T cell differentiation and TGF-β release. Thus, Gr1high monocytes may represent an interesting novel target for anti-fibrotic strategies.Matrine (Mat) is a kind of alkaloids isolated from Sophora flavescens Ait (Kushen). It has been found that Mat possesses a variety of pharmacological effect, including immunity-regulation activity, anti-inflammation. Animal and clinical studies revealed its anti-inflammatory and anti-fibrotic effects. However, the underlying mechanisms are not fully understood and the pharmacological activities of Mat are not potent enough. To obtain more potent candidates, we have synthesized a series of Mat derivatives via classical Michael addition and through screening for their biological activities, one of these derivatives, which we named it as M19, was found to exhibit more potent anti-inflammatory activity. Our animal study further revealed M19possesses a more potent anti-fibrotic effect than Mat.In the present study, we further explored the anti-hepatitic and anti-fibrotic effects of Mat and M19. Since Gr1high monocytes are recently demonstrated to play a crucial role in the development of hepatic fibrosis, we herein focused our study on the influence of Mat and M19on intrahepatic infiltration of Gr1high monocytes during acute and chronic hepatic inflammation.[Aim]Further elucidate the mechanisms underlying the anti-hepatitic and anti-fibrotic effects of Mat and M19, especially their influence on recruitment of Gr1high monocytes into injured liver.[Methods]1. Study on effects of Mat and M19on acute liver injury and hepatitisC57BL/6mice were injected intraperitoneally with CCl4(0.6ml/kg body weight). Mat (30mg/kg) or M19(30mg/kg) were administrated orally3h before CCl4injection. Serum were collected and analyzed for ALT levels24h after CCl4injection. Liver tissues were collected24h after CCl4injection. Hematoxylin-eosin staining of liver paraffin sections was performed to evaluate liver injury and mononuclear infiltration.2. Study on effects of Mat and M19on infiltration of Gr1high monocytes into injured liverLiver nonparenchymal cells (NPCs) was isolated from mouse livers of different experimental groups. The cellular composition of NPCs was analyzed by flow cytometry after staining with fluorescence-labeled antibodies against cell-surface markers of different immune cells. Immunohistochemistry examination of livers with anti-CD11b antibody was also performed to detect intrahepatic mononuclear infiltration. 3. Mechanism study for inhibition of Mat and M19on recruitment of Gr1high monocytes.In vivo effects of Mat and M19on the production of MCP-1was assessed by measuring MCP-1levels in sera of different experimental groups with ELISA, as well as intrahepatic MCP-1mRNA levels with real-time PCR. Immunohistochemistry examination of livers with anti-MCP-1antibody was also performed to detect intrahepatic MCP-1protein level. In vitro effects of Mat and M19on the production of MCP-1were assessed using LPS-stimulated NPCs and TNF-α-stimulated mouse HSC as cell models. MCP-1protein levels in supernatants of culture and intracellular mRNA levels were assayed with ELISA and rel-time PCR, respectively. The inhibitory effects of on chemotaxic activity of MCP-1were assay with transwell experiments.4. Study on anti-fibrotic effect of Mat and M19and their inhibition on recruitment of Gr1high monocytes into chronoc injured liver.C57BL/6mice were repeatedly injected intraperitoneally with CCl4(0.6ml/kg body weight,2times weekly for6weeks) to induced chronic liver injury. Mat (30mg/kg) or M19(30mg/kg) were administrated orally5times weekly from the first week. Collagen deposition was detected by histochemistry examination and liver hydroxyproline content measurement to evaluate the severity of hepatic fibrosis. To study the effects of Mat and M19on recruitment of Gr1high monocytes into chronic injured liver, NPCs was isolated from mouse livers of different experimental groups. The cellular composition of NPCs was analuzed by flow cytometry after staining with fluorescence-labeled antibodies against cell-surface markers of different immune cells. The intrahepatic MCP-1mRNA levels were assayed with real-time PCR. Immunohistochemistry examination of livers with anti-MCP-1antibody was also performed to detect intrahepatic MCP-1production.[Results]1. Mat and M19exhibited anti-inflammatory and protective effects against CCl4-induced acute liver injury.Single injection of CCl4(0.6ml/kg) resulted in periportal necroses with maximal damage at24hours, also reflected by highly elevated serum ALT activities. Toxic damage was accompanied by a considerable influx of leukocytes into the liver. Administration of Mat or M19(30mg/kg) significantly reduced CCl4-induced increase in serum ALT level, and also attenuated liver injury and intrahepatic infiltration of leukocytes. These results demonstrated the Mat and M19have anti-inflammatory and protective effects against CCl4-induced acute liver injury.2. Mat and M19blocked recruitment of Gr1high monocytes into injured liver.Single injection of CCl4(0.6ml/kg)resulted in significant increase in the total number of liver nonparenchymal cells (NPCs). Flow cytometry analysis of NPCs revealed that the percentage and total number of CD45+leukocytes were significantly increased at24hours after CCl4injection. Among these CD45-leukocytes, only percentage of CD11b+F4/80+Gr1high monocytes significantly increased, while the percentage of CD3+T cells decreased (possibly due to the increase of Gr1high monocytes) and those of other subpopulations remained unaltered. Further comparing the cell numbers of different subpopulations revealed that CDllb+F4/80+Gr1high subset massively increased after CCl4challenge, while other subsets only mildly increased or unaltered after CCl4injection and their relative contribution to intrahepatic leukocytes remained constant. These results confirmed that inflammatory Gr1high but not Grllow monocytes are massively recruited into the liver upon toxic injury. Administration of Mat or M19significantly reduced CCl4-induced increase in the total number of NPCs, as well as in the percentage and total number of CD45+leukocytes. Further flow cytometry analysis of cellular composition of NPCs from different experimental groups revealed that Mat and M19treatment resulted in significant decrease in the percentage and cell numbers of intrahepatic CD11b+F4/80+Gr1high monocytes, but not influence Grllow subset. Immunohistochemistry examination of livers with anti-MCP-1antibody revealed that the infiltrating cells at24hours after CCl4challenge, stained largely positive for the myeloid marker CD11b, and Mat and M19treatment remarkably attenuated the infiltration of CD11b+cells. In all of the above experiments, M19exhibited more potent inhibitory activities that Mat. These results demonstrated that Mat and M19blocked recruitment of Gr1high monocytes into injured liver.3. Mat and M19blocked recruitment of Grlhigh monocytes into injured liver through inhibiting production and chemotaxic activity of MCP-1.On CCl4-induced acute liver injury model, administration of Mat or M19(30mg/kg) significantly inhibited CCl4-induced increase in serum MCP-1levels and intrahepatic expression of MCP-1, and the inhibitory effects of M19were more potent than Mat. In in vitro experiment, M19inhibited LPS-stimulated release of MCP-1and TNF-a from mouse NPCs and TNF-α-stimulated expression of MCP-1in mouse HSCs dose-dependently in the range of5-50μM. Mat inhibited LPS-stimulated release of MCP-1from mouse NPCs and TNF-a-stimulated expression of MCP-1in mouse HSCs only at high dose of100μM. In transwell experiments, both M19and Mat inhibited MCP-1-stimulated migration of monocytes dose-dependently in the range of5-20μM, while M19exhibited a stronger inhibitory effect than Mat. These results demonstrated that Mat and M19blocked recruitment of Gr1high monocytes into injured liver through inhibiting production and chemotaxic activity of MCP-1.4. Mat and M19prevented development of hepatic fibrosis and blocked recruitment of Gr1high monocytes into chronic injured liver.Repeat injection of CCl4(0.6ml/kg body weight,2times weekly for6weeks) resulted in intrahepatic accumulation of collagen and increase in contents of liver hydroxyproline. Flow cytometry analysis of NPCs showed that CD11b+F4/80+Gr1high monocytes were also massively recruited into chronoc injured livers. Oral administration of Mat or M19(30mg/kg,5times weekly from the first week) significantly reduced the accumulation of collagen and contents of liver hydroxyproline, attenuated intrahepatic infiltration of Gr1high monocytes, and also inhibited intrahepatic expression of MCP-1. These results demonstrated that Mat and M19prevented development of hepatic fibrosis and blocked recruitment of Gr1high monocytes into chronic injured liver through inhibiting MCP-1production.[Conclusion]Mat and and its derivative M19exerts anti-hepatitic and anti-fibrotic effects through suppressing intrheptic production of MCP-1and inhibiting its chemotaxic activity, thus blocking the recruitment of Gr1high monocytes into injured liver. This finding not only extended our understanding of mechanisms underlying the anti-inflammatory and anti-fibrosic actions of M19and Mat, but also provides a basis for development of novel anti-fibrosic drugs.