Function Of Mouse Hepatic Stellate Cells-Derived Paracrine Signaling In Acute Liver Injury And Regeneration

Acute hepatic failure (AHF) is a severe liver injury with worldwide incidence, and causes multiorgan failure with an extremely high mortality rate, even if intensive care and drug treatment is provided. Liver transplantation has been shown to be the most effective therapy, but the procedure is limited by shortage of donor organs combined with the disadvantage of needing immunosuppressant treatment. Hepatocytes transplantation therapy and bioartificial liver support system application are also limited by shortage of seed cells and low efficiency of cell proliferation after cell transplantation. Therefore, management of severe AHF continues to be one of the most challenging problems in clinical medicine. Stem cell niche is specific living microenvironment for stem cells, maintain and regulate stem cell biological characteristics, and keep them in the balance of resting state, self-renewal and differentiation. Study the structure and function of hepatic stem cell niche will be propitious to fully stimulate the repair potential of endogenous stem cells in liver regeneration. This method neither need exogenous donor, nor has problem of immune rejection, and would be the ideal treatment of AHF. As one of the important supporting cells of liver stem cell niche, the role and mechanism of hepatic stellate cells (HSCs) has not been elucidated in liver regeneration. In this research, firstly we identified the role of activated HSCs in acute liver injury and regeneration; then established of isolation and culture model of mouse hepatic stellate cells, and observed the biological characteristics of different culture staged HSCs; finally identified whether the paracrine pathway of activated HSCs was involved in acute liver injury and regeneration, and clarified the relative mechanisms.Part I. The role of activated hepatic stellate cells in acute liver injury and regenerationObjective To confirm whether activated HSCs played significant role in promoting hepatocytes mitosis and/or hepatic stem cells proliferation in acute liver injury and regeneration.Method Acute liver injury was induced by APAP, which dissolved in warmed phosphate-buffered saline (PBS) at the dose of 300 mg/kg with intraperitoneal injection. For establishing depletion of activated HSCs model, we administered animals 3 doses of APAP plus gliotoxin (1 mg/kg body weight) by intervals of 3 days each dose, control animals received APAP plus DMSO alone. Cell toxicity of gliotoxin was observed on hepatocytes and Kupffer cells. To demonstrate that gliotoxin did not produce concomitant hepatocyte injury, we used APAP plus high dose of gliotoxin (3 mg/kg body weight) or APAP plus vehicle to treat animals respectively. Histological tissue sections and serum aminotransferase release were analyzed in both groups. Moreover, the data of aminotransferase release indicated no significant difference between the same detection time of two groups. Furthermore, cellular proliferation was detected by BrdU incorporation levels. And using the'loss of function'model of HSCs, through detecting the serum aminotransferase concentration, survival analysis, the degree of liver cell necrosis and apoptosis, infiltration of CD45-positive lymphocyte, proliferation of hepatocyte and oval cell, and other aspects to assess the role of activated HSCs in acute liver injury and regeneration.Result In the study of gliotoxin cell toxicity on hepatocyte, histological tissue scores and serum aminotransferase concentrations had no significant difference between gliotoxin-treated mice and control mice at any detected time points. In this 'loss of function'studies by depleting activated HSCs with gliotoxin. It was noteworthy that analysis of carbon particle incorporation in mice treated with APAP plus DMSO or APAP plus gliotoxin showed no differences. And the number of a-SMA-positive HSCs decreased significantly in gliotoxin-treated mice. A 90% decrease in the number of a-SMA-positive HSCs was observed after gliotoxin treatment (13.13±2.92)/FOV compared with (130.12±20.44)/FOV in control group (P<0.01). It also demonstrated that a significantly severe liver damage and declined survival rate were correlated with depletion of activated HSCs. The peak in liver damage was measured at 24 h after the final APAP treatment, both in control and gliotoxin-treated mice. However, the ALT and AST levels were increased by 73% (P<0.05) and 72%(P<0.01), respectively, in gliotoxin-treated mice compared with control mice. For survival analysis, a significant declined survival rate was observed for gliotoxin-treated animals. Only one animal died during the observation period in control mice, versus 55% of gliotoxin-treated mice (P<0.05). Semi-quantitative histological examination of liver tissue confirmed significant differences between the two groups. The average score in gliotoxin-treated group was (2.41±0.52) and (3.43±0.74) for vehicle-treated animals (P<0.01). A 47% increase in the number of infiltrating CD45-positive immune cells was observed after gliotoxin treatment (146.49±52.87)/FOV in treatment group versus (99.06±26.35)/FOV in control group (P<0.01). While few apoptotic cells were found in vehicle-treated hepatocytes. Quantification revealed a 3-fold increase in TUNEL positive hepatocyte-nuclei (16.94±9.33)/FOV when compared with control mice (4.29±3.06)/FOV (P<0.01). Furthermore, diminished HSCs activation resulted in a 66% decrease of the number of proliferating hepatocytes. This was accompanied by a dramatic decrease at the expression levels of 5 genes (OSM, EGF, IL6, HGF and SCF), ranging from 0.2 folds to 4.3 folds, which known to be up-regulated during hepatocyte replication. In particular, we found that depletion of activated HSCs inhibited oval cell reaction that was confirmed by decreased numbers of PanK-positive cells around the portal tracts and lowered gene expression level of cytokeratin 19 (CK19) in gliotoxin-treated liver. These data provide clear evidence that the activated HSCs are involved in both hepatocyte death and proliferation of hepatocytes and Oval cells in APAP-induced acute liver injury.Conclusion The current study showed that activated HSCs were required to attenuate acute liver injury, and enhanced liver regeneration by promoting both hepatocytes mitosis and hepatic stem cells proliferation.Part?. Establishment of isolation and culture model of mouse hepatic stellate cellsObjective To establish a high-performance and stable model for isolation and purification of the adult mouse hepatic stellate cells, and investigate the biological phenotype through primary culture and subculture.Method C57BL/6 mice were pretreated by intraperitoneal injection of methylene diphosphonic acid dichloride liposomes (CL2MDP-liposome; 10ml/kg body weight). Three days later, the liver was digested by in situ perfusion of pre-perfusion solution, 0.1% Pronase E and 0.075% Collagenase NB4G in turn. Continue to digest the liver using 0.02% DNase I liquid with magnetic stirring for 10 min in vitro, after low-speed centrifugation to remove residual hepatocytes, cells were treated with Optiprep density gradient solution to obtain the purified mouse HSCs. Trypan blue staining method was used to calculate cell production and viability. The cell purity was identified by mouse HSCs autofluorescence and oil red O staining. Using light microscopy, electronic transmission microscopy and double immunofluorescence staining of Desmin and a-SMA to observe morphological characteristics and biological phenotype of mouse HSCs.Result The number of C57BL/6 mice was 24, the average weight was (22.47±1.13) g, and the average liver weight was (1.30±0.14) g, the ratio of liver weight and body weight was 1:17.28. After CL2MDP-liposome pretreated, the yield rate of mouse HSCs was (1.62±0.34)×106 per gram of liver tissue, and cell purity was (94.44±1.89)%, and cell survival rate was (94.41±1.50)%. In PBS -liposome group, the yield rate of mouse HSCs was (1.37±0.23)×106/g liver, and cell purity was (90.18±1.61)%, and cell survival rate was (94.51±1.61)%. The cell yield rate and cell purity of CL2MDP-liposome group was better than PBS-liposome group in separately (P<0.05/P<0.01). The cell survival rate had no significant difference between two groups (P>0.05). The cell purity of two groups cells were close to 100% after the first cell passage. The activated mouse HSCs had different characteristics of submicroscopic structure and diversed express level of?-SMA.Conclusion CL2MDP-liposome could selectively removed Kupffer cells, and increased the yield rate and cell purity of mouse HSCs. This study established a stable model for isolation and purification mouse HSCs by improving digestive process and optimizing density gradient centrifugation. Early activated and fully activated mouse HSCs had different characteristics in cell phenotype and subcellular structure.Part III. The role of mouse HSCs-derived paracrine signaling in acute liver injury and regenerationObjective To confirm the function and mechanism of mouse hepatic stellate cells-derived paracrine signaling in acute liver injury and regeneration.Method Used the fifth day of primary cultured HSCs and the third generation of subcultured HSCs to prepare paracrine factors of the early activated HSCs (HSC-CM(5d)) and paracrine factors of the fully activated HSC (HSC-CM(P3)) respectively, then apply the protein chip to analysis the differences of protein composition between HSC-CM(5d) and HSC-CM(P3). Through the tail vein HSC-CM(5d) and HSC-CM(P3) were grafted respectively into the mice which were induced acute liver failure by APAP (intraperitoneal injection; 750mg/kg body weight), and observed the treatment effects of acute liver failure in vivo. Through examination of the promotion of cell proliferation, anti-cell necrosis and apoptosis, and regulating the level of inflammatory response to assess the role of HSCs-derived paracrine signaling. In addition, applied HSC-CM(5d) as conditioned medium to culture with hepatocytes in vitro, then through three aspects such as anti-APAP induced hepatocytes damage, promoting hepatocytes mitosis, and metabolic function to assess the function of HSC-CM(5d). Further isolated and cultured mouse adult hepatic progenitor cell (AHPC), used HSC-CM(5d) as conditioned medium to culture AHPC in vitro, and observed the impact of proliferation and metabolism of AHPC.ResultIn vivo study:Compared with control group, and HSC-CM (5d) group, the serum ALT levels in HSC-CM (P3) group decreased by 42% and 46%(P<0.01); AST levels decreased by 42% and 45%(P<0.01); and the ALT/AST levels had no significant difference between HSC-CM (P3) group and control group. Histological scores of acute liver injury suggested that HSC-CM (5d) group was (2.25±0.45), which was lower than HSC-CM (P3) group (3.51±0.57; P<0.01), and control group (3.75±0.51; P<0.05); no significant difference between HSC-CM (P3) group and control group. Survival analysis indicated that survival rate of HSC-CM (5d) mice, HSC-CM (P3) group, and control group were 85%,51%, and 42% in turn. HSC-CM (5d) had significantly superior survival rate than control group (P<0.05). There was no significant difference when compared HSC-CM (P3) group with neither HSC-CM (5d) group nor control group. A 64% decrease in the number of infiltrating CD45-positive immune cells was observed after HSC-CM (5d) treatment (45.58±28.72)/FOV in treatment group versus(125.20±54.05)/FOV in control group (P<0.01). After HSC-CM (5d) treatment, the serum concentrations of IFNy, ILlra, and IL1?were lower than control group; and TNFa had a 68% decrease (P<0.01); serum IL6 showed no significant changes between tow groups; but anti-inflammatory cytokine IL10 was significantly higher, about 1.9 times of control group (P<0.05). Quantification revealed a 83% decrease in TUNEL positive hepatocyte-nuclei (3.93±1.77) /FOV when compared with control mice (23.52±4.02) /FOV (P<0.01). Furthermore, HSC-CM (5d) treatment resulted in more than 2-folds increase in the number of proliferating hepatocytes. This was accompanied by a dramatic increase at the expression levels of 5 genes (OSM, EGF, IL6, HGF, and SCF), ranging from 2 folds to 32 folds, which known to be up-regulated during hepatocyte replication. In particular, we found that HSC-CM (5d) treatment promoted Oval cell reaction that was confirmed by increased numbers of Pank-positive cells around the portal tracts and highered gene expression level of CK19 and EpCAM in acute liver injury.Protein chip assay:Protein composition analysis showed that HSC-CM (5d) and HSC-CM (P3) expressed 69 cytokines of the total 144 detected factors. The overall composition of both was similar, which were mainly chemotactic factor, cytokines, growth factors, binding proteins, and extracellular matrix repair-related factors. We found that 7 kinds of cytokines between tow groups had a more than 2 times difference which were monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1?(MIP-1?), hepatocyte growth factor (HGF), interleukin 10 (IL10), matrix metalloproteinases-2 (MMPs-2), stem cell factor (SCF) and Fas ligand protein.In vitro study:APAP induced hepatocytes injury in vitro suggest that when APAP concentration was 1.25mmol/L, cell survival rate of HSC-CM (5%) group (86.17±4.84)% was better than HSC-CM (10%) group (78.01±5.10)%(P<0.05). When APAP concentration was 5mmol/L, cell survival rate of HSC-CM (5%) group was (45.67±2.23)%, higher than control group (42.33±2.58)%, and HSC-CM (10%) group (39.34±3.33)% respectively, (P<0.05). Using HSC-CM (5d) as conditioned medium cultured with hepatocytes, BrdU incorporation experiment suggested that the number of BrdU-Albumin-positive cells increased 47.97% in HSC-CM (5d) group (50.90±17.10) /FOV, when compared with control group (34.40±13.23)/FOV (P<0.05). In addition, detection of cellular metabolism suggested that Albumin and Urea concentration of culture supernatants was (6.56±2.32)?g/ml/d and (23.74±8.84)?g/ml/d, which were higher than control group (4.35±1.33)?g/ml/d and (13.44±6.12)?g/ml/d (P<0.05). Furthermore, using HSC-CM (5d) as conditioned medium cultured with AHPC in vitro. Compared with control group, HSC-CM (5d) group had three advantages, which were higher colony formation (29.18±3.71)%(P<0.05), a 66% increase in the number of BrdU-Albumin-positive cells (P<0.01), and increased Albumin and Urea concentration of culture supernatants (P<0.05/P<0.01).Conclusion Activated HSCs through the paracrine pathway involved in the process of acute liver injury and regeneration. Early activated and fully activated HSCs-derived paracrine factors play different roles in acute liver injury in vivo. Early activated HSCs-derived paracrine factors protected liver from APAP-induced damage and promoted liver regeneration, and the relative mechanisms included:anti-hepatocytes necrosis/apoptosis, promoting hepatocyte mitosis/hepatic stem cells proliferation, and down-regulating inflammation levels.Conclusion1. Activated HSCs attenuated acute liver injury and promoted hepatocyte mitosis/ hepatic stem cell proliferation.2. Early activated HSCs-derived paracrine factors protected liver from APAP induced damage and promoted liver regeneration.3. The relative mechanism included:anti-hepatocytes necrosis/apoptosis, promoting hepatocyte mitosis/hepatic stem cells proliferation, and down-regulating inflammation levels.